Low acidic species compositions and methods for producing and using the same

ABSTRACT

The instant invention relates to low acidic species (AR) compositions comprising a protein, e.g., an antibody, or antigen-binding portion thereof, and methods, e.g., cell culture and/or protein purification methods, for producing such low AR compositions. Methods for using such compositions to treat a disorder, e.g., a disorder in which TNFα is detrimental, are also provided.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/077,871, filed on Nov. 12, 2013, pending, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/893,068,filed on Oct. 18, 2013, the contents of each of which are herebyincorporated herein by reference in their entirety.

SEQUENCE LISTING

This specification incorporates by reference the Sequence Listingsubmitted via EFS web on Dec. 29, 2014 identified as 73904seqlist.txt,which is 10,923 bytes, and was created on Dec. 29, 2014. The SequenceListing, electronically filed, does not extend beyond the scope of thespecification and does not contain new matter.

BACKGROUND OF THE INVENTION

The production of compositions comprising proteins for biopharmaceuticalapplications involves the use of upstream process technologies (e.g.,cell culture) and downstream process technologies (e.g., proteinpurification) that are known to produce proteins exhibiting varyinglevels of protein variants and impurities within the composition. Suchprotein variants include, but are not limited to, the presence of acidicspecies, including process-related impurities. For example, inmonoclonal antibody (mAb) preparations, acidic species can be detectedby various methods, such as ion exchange chromatography, for example,WCX-10 HPLC (a weak cation exchange chromatography) or IEF (isoelectricfocusing). Because of their similar chemical characteristics to theantibody product molecules of interest, reduction of acidic species is achallenge in monoclonal antibody production.

Reduction of acidic species is particularly advantageous in the contextof commercially produced recombinant biotherapeutics, as they have thepotential to impact numerous product characteristics, including, but notlimited to, product stability, product safety and product efficacy.Accordingly, there remains a need in the art for low acidic speciescompositions and high-efficiency methods of producing proteincompositions, e.g., antibodies, having low levels of acidic species.

SUMMARY OF THE INVENTION

The present invention is based on the identification and optimization ofupstream and downstream process technologies for protein production,e.g., production of antibodies or antigen-binding portions thereof,resulting in the production of compositions comprising proteins thatcomprise low percentages of acidic species. As demonstrated herein,these low acidic species compositions have improved therapeutic efficacyand improved biological properties, for example, increased cartilagetissue penetration, reduced cartilage destruction, reduced synovialproliferation, reduced bone erosion, increased protection against thedevelopment of arthritis as measured by arthritic scores and/orhistopathology scores, reduced cell infiltration, reduced proteoglycanloss, reduced chondrocyte death, and/or increased TNFα affinity, ascompared to a non-low acidic species composition.

Accordingly, in one embodiment, the present invention provides a lowacidic species (low AR) composition comprising an antibody, orantigen-binding portion thereof, where the composition comprises about15% or less AR. In one aspect of this embodiment, the low AR compositioncomprises about 14% or less AR, 13% or less AR, 12% or less AR, 11% orless AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or less AR, 6%or less AR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5% or lessAR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9% or less AR, 1.8%or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR, 1.4% orless AR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1% or lessAR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% or less AR,0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or less AR, 0.1%or less AR, or 0.0% AR, and ranges within one or more of the preceding.In one aspect of this embodiment, the present invention provides a lowAR composition comprising an antibody, or antigen-binding portionthereof, where the composition comprises about 0.0% to about 10% AR,about 0.0% to about 5% AR, about 0.0% to about 4% AR, about 0.0% toabout 3% AR, about 0.0% to about 2% AR, about 3% to about 5% AR, about5% to about 8% AR, about 8% to about 10% AR, or about 10% to about 15%AR, and ranges within one or more of the preceding.

In one embodiment, the low AR composition comprises a first acidicspecies region (AR1) and a second acidic species region (AR2). In oneaspect of this embodiment, the low AR composition comprises about 0.1%or less AR1 and about 3% or less AR2, or about 0.0% AR1 and about 1.4%or less AR2. In a related embodiment, the low AR composition comprisesabout 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1,and ranges within one or more of the preceding. In one aspect of thisembodiment, the present invention provides a low AR compositioncomprising an antibody, or antigen-binding portion thereof, where thecomposition comprises about 0.0% to about 10% AR1, about 0.0% to about5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% AR1, about0.0% to about 2% AR1, about 3% to about 5% AR1, about 5% to about 8%AR1, or about 8% to about 10% AR1, or about 10% to about 15% AR1, andranges within one or more of the preceding.

In one aspect of this embodiment, the low AR composition comprises about15% or less AR2, 14% or less AR2, 13% or less AR2, 12% or less AR2, 11%or less AR2, 10% or less AR2, 9% or less AR2, 8% or less AR2, 7% or lessAR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2, 4% or less AR2,3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2% or less AR2, 1.9%or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5%or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1%or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% orless AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% orless AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and rangeswithin one or more of the preceding. In one aspect of this embodiment,the present invention provides a low AR composition comprising anantibody, or antigen-binding portion thereof, where the compositioncomprises about 0.0% to about 10% AR2, about 0.0% to about 5% AR2, about0.0% to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% to about 2%AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, or about 8% toabout 10% AR2, or about 10% to about 15% AR2, and ranges within one ormore of the preceding.

In another embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab, comprises about 1.4% or less AR. For example,in one aspect of this embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab comprising about 1.4% or less AR can compriseabout 0.0% AR1 and about 1.4% or less AR2.

In another aspect, the present invention provides compositionscomprising an antibody, or antigen-binding portion thereof, wherein thecomposition is substantially free of acidic species and otherprocess-related impurities, including, for example, host cell proteins(HCPs), host nucleic acids, chromatographic materials, and/or mediacomponents, as well as product related impurities such as aggregates.

In one embodiment, the antibody, or antigen-binding portion thereof, ofthe compositions disclosed herein is an anti-TNFα antibody, orantigen-binding portion thereof. For example, in one aspect of thisembodiment, the anti-TNFα antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(d) of about 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ S⁻¹ or less. In another aspect of thisembodiment, the anti-TNFα antibody, or antigen-binding portion thereof,comprises a light chain variable region (LCVR) having a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3; and a heavy chainvariable region (HCVR) having a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4.

In still another aspect of this embodiment, the anti-TNFα antibody, orantigen-binding portion thereof, comprises a light chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 2. In yet another aspect of this embodiment, the anti-TNFαantibody, or antigen-binding portion thereof, is adalimumab, or anantigen binding-portion thereof.

In one embodiment, the low AR composition of the invention comprisesadalimumab, and has a percentage of AR that is not the same as thepercentage of AR present in adalimumab formulated as HUMIRA® ascurrently approved and described in the “Highlights of PrescribingInformation” for HUMIRA® (adalimumab) Injection (Revised January 2008),the contents of which are hereby incorporated herein by reference.

In another embodiment, the low AR composition of the invention comprisesadalimumab, and has a percentage of AR that is lower than the percentageof AR present in adalimumab formulated as HUMIRA® as currently approvedand described in the “Highlights of Prescribing Information” for HUMIRA®(adalimumab) Injection (Revised January 2008), the contents of which arehereby incorporated herein by reference.

In another embodiment, the present invention provides low ARcompositions comprising an anti-TNFα antibody, or antigen-bindingportion thereof, comprising a light chain variable region (LCVR) havinga CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3; and a heavychain variable region (HCVR) having a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 6, and a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, wherein the composition comprises less thanabout 10% AR. In one aspect of this embodiment, the anti-TNFα antibody,or antigen-binding portion thereof, comprises a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 1 anda heavy chain variable region comprising the amino acid sequence setforth in SEQ ID NO: 2, wherein the composition comprises less than about10% AR. In another aspect of this embodiment, the anti-TNFα antibody, orantigen-binding portion thereof, is adalimumab, or an antigenbinding-portion thereof, and the composition comprises less than about10% AR. In one aspect of this embodiment, the low AR compositioncomprising an anti-TNFα antibody, or antigen-binding portion thereof,comprises about 0.1% or less AR1 and about 3% or less AR2, or about 0.0%AR1 and about 1.4% or less AR2.

In one embodiment, the acidic species in the low AR compositioncomprising an antibody, or antigen-binding portion thereof (e.g., ananti-TNFα antibody, or antigen binding portion thereof, such asadalimumab) comprise one or more variants selected from the groupconsisting of charge variants, structure variants and fragmentationvariants (see, for example, FIG. 188).

For example, in one aspect of this embodiment, the charge variants inthe low AR composition are AR1 species and comprise, for example,deamidation variants, glycation variants, afucosylation variants,methylglyoxal (MGO) variants or citric acid variants. For example, whenthe low AR composition comprises adalimumab, the deamidation variantscan result from deamidation occurring at asparagine residues comprisingAsn393 and Asn329 of adalimumab and at glutamine residues comprisingGln3 and Gln6. In another aspect of this embodiment, when the low ARcomposition comprises adalimumab, the glycation variants can result fromglycation occurring at Lys98 and Lys151 of adalimumab.

In another aspect of this embodiment, the structure variants in the lowAR composition comprising an antibody, or antigen-binding portionthereof (e.g., an anti-TNFα antibody, or antigen binding portionthereof, such as adalimumab) are AR1 species and comprise, for example,glycosylation variants or acetonation variants.

In still another aspect of this embodiment, the fragmentation variantsin the low AR composition comprising an antibody, or antigen-bindingportion thereof (e.g., an anti-TNFα antibody, or antigen binding portionthereof, such as adalimumab), are AR1 species and comprise, for example,Fab fragment variants, C-terminal truncation variants or variantsmissing a heavy chain variable domain.

In another embodiment, the acidic species in the low AR compositioncomprising an antibody, or antigen-binding portion thereof (e.g., ananti-TNFα antibody, or antigen binding portion thereof, such asadalimumab), are AR2 species, and comprise charge variants, such asdeamidation variants or glycation variants. For example, when the low ARcomposition comprises adalimumab, the deamidation variants can resultfrom deamidation occurring at asparagine residues comprising Asn393 andAsn329 of adalimumab and at glutamine residues comprising Gln3 and Gln6.In another aspect of this embodiment, when the low AR compositioncomprises adalimumab, the glycation variants result from glycationoccurring at Lys98 and Lys151 of adalimumab.

In one embodiment, the percent of acidic species in a low AR compositionis determined using ion exchange chromatography, for example WCX-10HPLC. In another aspect of this embodiment, the percent acidic speciesin a low AR composition is determined using isoelectric focusing (IEF).

In one embodiment, the low AR compositions of the invention compriseproduct preparation-derived acidic species. For example, in one aspectof this embodiment, the acidic species are cell culture-derived acidicspecies. In another aspect of this embodiment, the acidic species of thelow AR compositions are storage-derived acidic species which areprimarily generated when stored under process, intermediate or shelfstorage conditions prior to use.

In still another embodiment, the invention provides low AR compositionsthat further comprise a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods for treating asubject having a disorder in which TNFα is detrimental, by administeringto the subject a low AR composition of the invention, e.g., a low ARadalimumab composition, thereby treating the subject having a disorderin which TNFα is detrimental. In one aspect of this embodiment, thedisorder in which TNFα is detrimental is selected from the groupconsisting of rheumatoid arthritis (RA), psoriasis, psoriatic arthritis,ankylosing spondylitis, juvenile idiopathic arthritis (JIA), ulcerativecolitis, and Crohn's Disease.

In one aspect, upstream methods for producing the low AR compositions ofthe invention are included. In one embodiment, a method for producing alow acidic species composition comprising an antibody, or antigenbinding portion thereof, comprises culturing cells expressing theantibody, or antigen binding portion thereof, in a cell culture mediacomprising an increased concentration of an amino acid selected from thegroup consisting of arginine, lysine, ornithine and histidine, or acombination thereof, as compared to the amino acid concentration in cellculture media used to produce a non-low acidic species compositioncomprising the antibody, or antigen binding portion thereof. In anotheraspect of this embodiment, the amino acid concentration in the culturemedia is between about 0.025 and 20 g/L.

In another embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises culturing cells expressing the antibody, or antigen bindingportion thereof, in a cell culture media comprising an increasedconcentration of calcium as compared to the calcium concentration incell culture media used to produce a non-low acidic species compositioncomprising the antibody, or antigen binding portion thereof. In oneaspect of this embodiment, the calcium concentration is between about0.005 and 5 mM. In another aspect of this embodiment, the cell culturemedia further comprises an increased concentration of an amino acidselected from the group consisting of arginine, lysine, ornithine andhistidine, or a combination thereof, as compared to the amino acidconcentration in cell culture media used to produce a non-low acidicspecies composition comprising the antibody, or antigen binding portionthereof.

In still another embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises culturing cells expressing the antibody, or antigen bindingportion thereof, in a cell culture media comprising an increasedconcentration of niacinamide, calcium, and at least one amino acid, ascompared to the concentration of niacinamide, calcium, and amino acid inthe cell culture media used to produce a non-low acidic speciescomposition comprising the antibody, or antigen binding portion thereof.In one aspect of the embodiment, the at least one amino acid is selectedfrom the group consisting of arginine, lysine, ornithine and histidine,and combinations thereof.

In yet another embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises culturing cells expressing the antibody, or antigen bindingportion thereof, in a cell culture media having a pH of between about7.1-about 6.8.

In still another embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises culturing cells expressing the antibody, or antigen bindingportion thereof, in a cell culture media having an altered exchange rateas compared to the exchange rate of cell culture media used to produce anon-low acidic species composition comprising the antibody, or antigenbinding portion thereof.

In another embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises culturing cells expressing the antibody, or antigen bindingportion thereof, extracting a clarified harvest from the cell culture,and adding one or more amino acids to the clarified harvest. In oneaspect of this embodiment, the one or more amino acids are selected fromthe group consisting of arginine, histidine, lysine, aspartic acid,glutamic acid and leucine, and combinations thereof.

In yet another embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises culturing cells expressing the antibody, or antigen bindingportion thereof, extracting a clarified harvest from the cell culture,and adjusting the pH of the clarified harvest to between about 4.5 and6.5.

In another aspect of the invention, upstream methods for producing thelow AR compositions of the invention are included. In one embodiment,the invention includes a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprising contacting a first sample comprising the antibody, or antigenbinding portion thereof, to a chromatography media, wherein the contactoccurs in the context of a loading buffer; washing the chromatographymedia with a wash buffer that is substantially the same as the loadingbuffer; and collecting a chromatography sample, wherein thechromatography sample comprises a composition of the antibody, orantigen binding portion thereof, which contains less than about 10%acidic species, thereby producing a low acidic species compositioncomprising an antibody, or antigen binding portion thereof. In oneaspect of this embodiment, the bound antibody material is eluted with abuffer having a different composition than the wash buffer. In anotheraspect of this embodiment, the chromatography media is selected from thegroup consisting of anion exchange adsorbent material, cation exchangeadsorbent material, mixed mode media, cation exchange mixed mode media,and anion exchange mixed mode media. In one embodiment, thechromatography media is a mixed mode media comprising cation exchange(CEX) and hydrophobic interaction functional groups. In anotherembodiment, the chromatography media is a mixed mode media comprisinganion exchange (AEX) and hydrophobic interaction functional groups. Forexample, the mixed mode media may be Capto MMC resin, the CEX resin maybe the Poros XS resin, and the AEX resin may be the Poros 50HQ resin.

In one embodiment, the chromatography media is a CEX adsorbent materialor a mixed mode media, and the pH of the loading and wash buffers islower than the isoelectric point of the antibody. In another embodiment,the chromatography sample contains a reduced level of antibody fragmentsas compared to the first sample. In still another embodiment, thechromatography sample contains a reduced level of host cell proteins ascompared to the first sample. In yet another embodiment, thechromatography sample contains a reduced level of one or more of chargevariants (e.g., deamidation variants, glycation variants, afucosylationvariants, MGO variants or citric acid variants), structure variants(e.g., glycosylation variants or acetonation variants), or fragmentationvariants (e.g., Fab fragment variants, C-terminal truncation variants orvariants missing a heavy chain variable domain) as compared to the firstsample.

In one embodiment, a method for producing a low acidic speciescomposition comprising an antibody, or antigen binding portion thereof,comprises contacting a first sample comprising the antibody, or antigenbinding portion thereof, to an affinity chromatography media (e.g., aProtein A resin) in a load buffer, and eluting said sample from theaffinity chromatography media as a first eluted sample; contacting thefirst eluted sample to an anion exchange (AEX) chromatography adsorbentmaterial (e.g., a Poros 50HQ resin) in a load buffer, and eluting saidsample from the AEX chromatography adsorbent material as a second elutedsample; and contacting the second eluted sample to a cation exchange(CEX) chromatography adsorbent material (e.g., a Poros XS resin) in aload buffer, and eluting said sample from the CEX chromatographyadsorbent material as a third eluted sample, wherein the third elutedsample comprises a composition of the antibody, or antigen bindingportion thereof, which contains less than about 3% acidic species,thereby producing a low acidic species composition comprising anantibody, or antigen binding portion thereof. In one embodiment, thesecond eluted sample is contacted to a CEX chromatography at least oneadditional time. In one embodiment, the method further comprisesperforming viral filtration on the third eluted sample resulting in afiltered sample. In another embodiment, the method further comprisesfiltering the filtered sample using ultrafiltration/diafilteration(UF/DF).

In another aspect, the invention provides a method for producing a lowacidic species composition comprising an antibody, or antigen bindingportion thereof, the method comprising contacting a sample comprising anantibody, or antigen binding portion thereof to one or more of the groupconsisting of: an anion exchange (AEX) chromatography adsorbentmaterial, a cation exchange (CEX) chromatography adsorbent material, amixed mode media, a cation exchange mixed mode media, and an anionexchange mixed mode media, in a load buffer, and eluting the sample fromthe AEX chromatography adsorbent material, the CEX chromatographyadsorbent material, the mixed mode media, the cation exchange mixed modemedia, or the anion exchange mixed mode media, wherein the eluted samplecomprises a composition of the antibody, or antigen binding portionthereof, which contains less than about 3% acidic species, therebyproducing a low acidic species composition comprising an antibody, orantigen binding portion thereof. In one aspect of this embodiment, themethod further comprises contacting the eluted sample to a hydrophobicinteraction chromatography (HIC) media.

The present invention is further illustrated by the following detaileddescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of total arginine concentration in adalimumabproducing cell line 2, media 1 on viable cell density (n=2).

FIG. 2 depicts the effect of total arginine concentration in adalimumabproducing cell line 2, media 1 on viability (n=2).

FIG. 3 depicts the effect of total arginine concentration in adalimumabproducing cell line 2, media 1 on harvest titer (n=2).

FIG. 4 depicts the effect of total arginine concentration in adalimumabproducing cell line 2, media 1 on day 10 WCX-10 profile total acidicregions (n=2).

FIG. 5 depicts the effect of total arginine concentration in adalimumabproducing cell line 2, media 1 on day 12 WCX-10 profile total acidicregions (n=2).

FIG. 6 depicts the effect of total arginine concentration in adalimumabproducing cell line 3, media 1 on viable cell density (n=2).

FIG. 7 depicts the effect of total arginine concentration in adalimumabproducing cell line 3, media 1 on viability (n=2).

FIG. 8 depicts the effect of total arginine concentration in adalimumabproducing cell line 3, media 1 on harvest titer (n=2).

FIG. 9 depicts the effect of total arginine concentration in adalimumabproducing cell line 3, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 10 depicts the effect of total arginine concentration in adalimumabproducing cell line 1, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 11 depicts the effect of arginine addition to adalimumab producingcell line 1, media 2 on day 11 on WCX-10 profile total acidic regions(n=2).

FIG. 12 depicts the effect of arginine addition to adalimumab producingcell line 2, media 3 on WCX-10 profile total acidic regions (n=2).

FIG. 13 depicts the effect of total arginine concentration in mAb1producing cell line on WCX-10 profile total acidic regions (n=1).

FIG. 14 depicts the effect of total arginine concentration in mAb2producing cell line on WCX-10 profile total acidic regions (n=2)

FIG. 15 depicts the effect of carboxypeptidase digestion of product fromadalimumab producing cell line 3, media 1 experiment on WCX-10 profiletotal acidic regions (n=1).

FIG. 16 depicts the effect of carboxypeptidase digestions of productfrom mAb2 producing cell line on WCX-10 profile total acidic regions(n=2).

FIG. 17 depicts the effect of total lysine concentration in adalimumabproducing cell line 2, media 1 on viable cell density (n=2).

FIG. 18 depicts the effect of total lysine concentration in adalimumabproducing cell line 2, media 1 on viability (n=2).

FIG. 19 depicts the effect of total lysine concentration in adalimumabproducing cell line 2, media 1 on harvest titer (n=2).

FIG. 20 depicts the effect of total lysine concentration in adalimumabproducing cell line 2, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 21 depicts the effect of total lysine concentration in adalimumabproducing cell line 3, media 1 on viable cell density (n=2).

FIG. 22 depicts the effect of total lysine concentration in adalimumabproducing cell line 3, media 1 on viability (n=2).

FIG. 23 depicts the effect of total lysine concentration in adalimumabproducing cell line 3, media 1 on harvest titer (n=2).

FIG. 24 depicts the effect of total lysine concentration in adalimumabproducing cell line 3, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 25 depicts the effect of total lysine concentration in adalimumabproducing cell line 1, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 26 depicts the effect of lysine addition to adalimumab producingcell line 1, media 2 on WCX-10 profile total acidic regions (n=2).

FIG. 27 depicts the effect of lysine addition to adalimumab producingcell line 2, media 3 on WCX-10 profile total acidic regions (n=2).

FIG. 28 depicts the effect of total lysine concentration in mAb1producing cell line on WCX-10 profile total acidic regions (n=1).

FIG. 29 depicts the effect of total lysine concentration in mAb2producing cell line on WCX-10 profile total acidic regions (n=2).

FIG. 30 depicts the effect of carboxypeptidase digestion of product fromcell line 3, media 1 experiment on WCX-10 profile total acidic regions(n=1).

FIG. 31 depicts the effect of carboxypeptidase digestions of productfrom mAb2 producing cell line on WCX-10 profile total acidic regions(n=2).

FIG. 32 depicts the effect of total histidine concentration inadalimumab producing cell line 2, media 1 on viable cell density (n=2).

FIG. 33 depicts the effect of total histidine concentration inadalimumab producing cell line 2, media 1 on viability (n=2).

FIG. 34 depicts the effect of total histidine concentration inadalimumab producing cell line 2, media 1 on harvest titer (n=2).

FIG. 35 depicts the effect of total histidine concentration inadalimumab producing cell line 2, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 36 depicts the effect of total histidine concentration inadalimumab producing cell line 3, media 1 on viable cell density (n=2).

FIG. 37 depicts the effect of total histidine concentration inadalimumab producing cell line 3, media 1 on viability (n=2).

FIG. 38 depicts the effect of total histidine concentration inadalimumab producing cell line 3, media 1 on harvest titer (n=2).

FIG. 39 depicts the effect of total histidine concentration inadalimumab producing cell line 3, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 40 depicts the effect of total histidine concentration inadalimumab producing cell line 1, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 41 depicts the effect of histidine addition to adalimumab producingcell line 1, media 2 on WCX-10 profile total acidic regions (n=2).

FIG. 42 depicts the effect of histidine addition to adalimumab producingcell line 2, media 3 on WCX-10 profile total acidic regions (n=2).

FIG. 43 depicts the effect of total histidine concentration in mAb1producing cell line on WCX-10 profile total acidic regions (n=1).

FIG. 44 depicts the effect of total histidine concentration in mAb2producing cell line on WCX-10 profile total acidic regions (n=2).

FIG. 45 depicts the effect of carboxypeptidase digestion of product fromcell line 3, media 1 experiment on WCX-10 profile total acidic regions(n=1).

FIG. 46 depicts the effect of carboxypeptidase digestions of productfrom mAb2 producing cell line on WCX-10 profile total acidic regions(n=2).

FIG. 47 depicts the effect of total ornithine concentration inadalimumab producing cell line 2, media 1 on viable cell density (n=2).

FIG. 48 depicts the effect of total ornithine concentration inadalimumab producing cell line 2, media 1 on viability (n=2).

FIG. 49 depicts the effect of total ornithine concentration inadalimumab producing cell line 2, media 1 on harvest titer (n=2).

FIG. 50 depicts the effect of total ornithine concentration inadalimumab producing cell line 2, media 1 on WCX-10 profile total acidicregions.

FIG. 51 depicts the effect of total ornithine concentration inadalimumab producing cell line 3, media 1 on viable cell density (n=2).

FIG. 52 depicts the effect of total ornithine concentration inadalimumab producing cell line 3, media 1 on viability (n=2).

FIG. 53 depicts the effect of total ornithine concentration inadalimumab producing cell line 3, media 1 on harvest titer (n=2).

FIG. 54 depicts the effect of total ornithine concentration inadalimumab producing cell line 3, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 55 depicts the effect of total ornithine concentration inadalimumab producing cell line 1, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 56 depicts the effect of ornithine addition to adalimumab producingcell line 1, media 2 on WCX-10 profile total acidic regions (n=2).

FIG. 57 depicts the effect of ornithine addition to adalimumab producingcell line 2, media 3 on WCX-10 profile total acidic regions (n=2).

FIG. 58 depicts the effect of total ornithine concentration in mAb1producing cell line on WCX-10 profile total acidic regions (n=1).

FIG. 59 depicts the effect of total ornithine concentration in mAb2producing cell line on WCX-10 profile total acidic regions (n=2).

FIG. 60 depicts the effect of carboxypeptidase digestion of product fromcell line 3, media 1 experiment on WCX-10 profile total acidic regions(n=1).

FIG. 61 depicts the effect of carboxypeptidase digestions of productfrom mAb2 producing cell line on WCX-10 profile total acidic regions(n=2).

FIG. 62 depicts the effect of multiple amino acid additions toadalimumab producing cell line 2, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 63 depicts the effect of increased arginine and lysineconcentration in adalimumab producing cell line 1, media 1 on viablecell density (n=3).

FIG. 64 depicts the effect of increased arginine and lysineconcentration in adalimumab producing cell line 1, media 1 on viability(n=3).

FIG. 65 depicts the effect of increased arginine and lysineconcentration in adalimumab producing cell line 1, media 1 on culturetiter (n=3).

FIG. 66 depicts the effect of increased arginine and lysineconcentration in adalimumab producing cell line 1, media 1 on WCX-10profile total acidic regions (n=2).

FIG. 67 depicts the effect of arginine, lysine and pH modulation toadalimumab producing cell line 1, media 1 on viable cell density (n=2).

FIG. 68 depicts the effect of arginine, lysine and pH modulation toadalimumab producing cell line 1, media 1 on viability (n=2).

FIG. 69 depicts the effect of arginine, lysine and pH modulation toadalimumab producing cell line 1, media 1 on culture titer (n=2).

FIG. 70 depicts the effect of arginine, lysine and pH modulation toadalimumab producing cell line 1, media 1 on WCX-10 profile total acidicregions (n=2).

FIG. 71 depicts the effect of total calcium concentration in adalimumabproducing cell line 2, media 1 on viable cell density (n=2).

FIG. 72 depicts the effect of total calcium concentration in adalimumabproducing cell line 2, media 1 on viability (n=2).

FIG. 73 depicts the effect of total calcium concentration in adalimumabproducing cell line 2, media 1 on harvest titer (n=2).

FIG. 74 depicts the effect of total calcium concentration in adalimumabproducing cell line 2, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 75 depicts the effect of total calcium concentration in adalimumabproducing cell line 3, media 1 on viable cell density (n=2).

FIG. 76 depicts the effect of total calcium concentration in adalimumabproducing cell line 3, media 1 on viability (n=2).

FIG. 77 depicts the effect of total calcium concentration in adalimumabproducing cell line 3, media 1 on harvest titer (n=2)

FIG. 78 depicts the effect of total calcium concentration in adalimumabproducing cell line 3, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 79 depicts the effect of total calcium concentration in adalimumabproducing cell line 1, media 1 on WCX-10 profile total acidic regions(n=2).

FIG. 80 depicts the effect of calcium addition to adalimumab producingcell line 1, media 2 on WCX-10 profile total acidic regions (n=2).

FIG. 81 depicts the effect of calcium addition to adalimumab producingcell line 2, media 3 on WCX-10 profile total acidic regions (n=2).

FIG. 82 depicts the effect of total calcium concentration in mAb1producing cell line on WCX-10 profile total acidic regions (n=2).

FIG. 83 depicts the effect of total calcium concentration in mAb2producing cell line on WCX-10 profile total acidic regions (n=2).

FIGS. 84A-B depict the effect of multiple amino acid additions to cellline 1, media 1 on WCX-10 profile total acidic regions a) overallprediction plot, b) prediction plots for each additive (n=2).

FIG. 85 depicts the effect of niacinamide addition to adalimumabproducing cell line 1, media 1 on viable cell density (n=2).

FIG. 86 depicts the effect of niacinamide addition to adalimumabproducing cell line 1, media 1 on viability (n=2).

FIG. 87 depicts the effect of niacinamide addition to adalimumabproducing cell line 1, media 1 on harvest titer (n=2).

FIG. 88 depicts the effect of niacinamide addition to adalimumabproducing cell line 1, media 1 on Day 11 WCX-10 profile total acidicregions (n=2).

FIG. 89 depicts the effect of niacinamide addition to adalimumabproducing cell line 1, media 1 on Day 12 WCX-10 profile total acidicregions (n=2).

FIG. 90 depicts the effect of niacinamide addition to mAb2 producingcell line, media 1 on viable cell density (n=2).

FIG. 91 depicts the effect of niacinamide addition to mAb2 producingcell line, media 1 on viability (n=2).

FIG. 92 depicts the effect of niacinamide addition to mAb2 producingcell line, media 1 on harvest titer (n=2).

FIG. 93 depicts the effect of niacinamide addition to mAb2 producingcell line, media 1 on WCX-10 profile total acidic regions (n=2).

FIGS. 94A-D depict the effect of amino acid supplementation to CD mediaGIA-1 in adalimumab-producing CHO cell line #1 on (A) culture growth,(B) culture viability, (C) acidic species, and (D) MGO modification.

FIG. 95 depicts the effect of pH modulation of adalimumab producing cellline 1, media 1 on viable cell density.

FIG. 96 depicts the effect of pH modulation of adalimumab producing cellline 1, media 1 on viability.

FIG. 97 depicts the effect of pH modulation of adalimumab producing cellline 1, media 1 on harvest titer.

FIG. 98 depicts the effect of pH modulation of adalimumab producing cellline 1, media 1 on WCX-10 profile total acidic regions.

FIG. 99 depicts the effect of pH modulation of adalimumab producing cellline 1, media 2 on viable cell density.

FIG. 100 depicts the effect of pH modulation addition of adalimumabproducing adalimumab producing cell line 1, media 2 on viability.

FIG. 101 depicts the effect of pH modulation of adalimumab producingcell line 1, media 2 on harvest titer.

FIG. 102 depicts the effect of pH modulation of adalimumab producingcell line 1, media 2 on WCX-10 profile total acidic regions.

FIG. 103 depicts the effect of pH modulation of adalimumab producingcell line 3, media 1 on viable cell density.

FIG. 104 depicts the effect of pH modulation adalimumab producing cellline 3, media 1 on viability.

FIG. 105 depicts the effect of pH modulation of adalimumab producingcell line 3, media 1 on harvest titer.

FIG. 106 depicts the effect of pH modulation of adalimumab producingcell line 3, media 1 on WCX-10 profile total acidic regions.

FIG. 107 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 35° C. on viable cell density.

FIG. 108 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 35° C. on viability.

FIG. 109 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 35° C. on harvest titer.

FIG. 110 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 35° C. on WCX-10 profile total acidicregions.

FIG. 111 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 33° C. on viable cell density.

FIG. 112 depicts the effect of dissolved oxygen modulation to adalimumabproducing cell line 1, media 2 at 33° C. on viability.

FIG. 113 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 33° C. on harvest titer.

FIG. 114 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 2 at 33° C. on WCX-10 profile total acidicregions.

FIG. 115 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 1 at 35° C. on viable cell density.

FIG. 116 depicts the effect of dissolved oxygen modulation to adalimumabproducing cell line 1, media 1 at 35° C. on viability.

FIG. 117 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 1 at 35° C. on harvest titer.

FIG. 118 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 1, media 1 on WCX-10 profile total acidic regions.

FIG. 119 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 3, media 1 on viable cell density.

FIG. 120 depicts the effect of dissolved oxygen modulation to adalimumabproducing cell line 3, media 1 on viability.

FIG. 121 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 3, media 1 on harvest titer.

FIG. 122 depicts the effect of dissolved oxygen modulation of adalimumabproducing cell line 3, media 1 on WCX-10 profile total acidic regions.

FIG. 123 depicts the effect of dissolved oxygen modulation to mAb2producing cell line, media 1 on viable cell density.

FIG. 124 depicts the effect of dissolved oxygen modulation addition tomAb2 producing cell line, media 1 on viability.

FIG. 125 depicts the effect of dissolved oxygen modulation to mAb2producing cell line, media 1 on harvest titer.

FIG. 126 depicts the effect of dissolved oxygen modulation to mAb2producing cell line, media 1 on WCX-10 profile total acidic regions.

FIG. 127 depicts an acidification sample preparation scheme.

FIG. 128 depicts an arginine sample preparation scheme.

FIG. 129 depicts a histidine sample preparation scheme.

FIG. 130 depicts a lysine sample preparation scheme.

FIG. 131 depicts a methionine sample preparation scheme.

FIG. 132 depicts an amino acid sample preparation scheme.

FIG. 133 depicts a CDM clarified harvest sample preparation scheme.

FIG. 134 depicts an acid-type pH study sample preparation scheme.

FIG. 135 depicts the effect of low pH treatment with subsequentneutralization on initial acidic variant content.

FIG. 136 depicts the effect of low pH treatment with subsequentneutralization on acidic variant formation rate.

FIG. 137 depicts the effect of sample preparation method on initialacidic variant content.

FIG. 138 depicts the effect of sample preparation method on initialacidic variant content.

FIG. 139 depicts the dose dependent effect of arginine on reduction ofacidic variant formation rate.

FIG. 140 depicts the effect of histidine concentration on initial acidicvariant content.

FIG. 141 depicts the effect of histidine concentration on acidic variantformation rate.

FIG. 142 depicts the effect of lysine on initial acid variant content.

FIG. 143 depicts the effect of lysine on acidic variant formation rate.

FIG. 144 depicts the effect of methionine on initial acid variantcontent.

FIG. 145 depicts the effect of methionine on acidic variant formationrate.

FIG. 146 depicts the effect of amino acids on initial acid variantcontent.

FIG. 147 depicts the effect of amino acids on acidic variant formationrate.

FIG. 148 depicts the effect of alternative additives on initial acidvariant content.

FIG. 149 depicts the effect of alternative additives on acidic variantformation rate.

FIG. 150 depicts the effect of low pH/arginine treatment on adalimumabCDM initial acid variant content.

FIG. 151 depicts the effect of low pH/arginine treatment on adalimumabCDM acidic variant formation rate.

FIG. 152 depicts the effect of low pH/arginine treatment on mAb Bhydrolysate initial acid variant content.

FIG. 153 depicts the effect of low pH/arginine treatment on mAb Bhydrolysate acidic variant formation rate.

FIG. 154 depicts the effect of low pH/arginine treatment on mAb Chydrolysate initial acid variant content.

FIG. 155 depicts the effect of low pH/arginine treatment on mAb Chydrolysate acidic variant formation rate.

FIG. 156 depicts the effect of acid type/pH on acid variant content.

FIG. 157 depicts the effect of acid concentration on acid variantcontent.

FIG. 158 depicts the effect of acid concentration on acid variantcontent.

FIG. 159 depicts the effect of neutralization on acid variant content.

FIG. 160 depicts the effect of neutralization on acid variant content.

FIG. 161 depicts the effect of medium exchange rate and thesupplementation of amino acids arginine and lysine on total acidicspecies reduction.

FIG. 162 depicts LC/MS peptide mapping analysis of exemplary antibodiesexpressed in the context of the cell culture conditions of the instantinvention, including preparation of specific mass traces for bothmodified and non-modified peptides in order to accurately quantify thetotal amount of MGO modification. Mass spectra are also analyzed for thespecific region of the chromatogram to confirm the peptide identity.

FIG. 163 depicts a chromatogram wherein the total acidic speciesassociated with the expression of adalimumab is divided into a firstacidic species region (AR1) and a second acidic species region (AR2).

FIG. 164 depicts the AR growth at 25° C. of low and high AR containingsamples.

FIG. 165 depicts a process chromatogram of pH gradient elution in thecontext of AEX chromatography.

FIG. 166 depicts a process chromatogram of a linear gradient elution byincreasing anion concentration in the context of AEX chromatography.

FIG. 167 depicts a process chromatogram of fractionation of 300 g/L loadand wash in the context of AEX chromatography.

FIG. 168 depicts the effect of pH on AR reduction in the context of AEXchromatography.

FIG. 169 depicts a process chromatogram at different salt (cation)concentrations in the context of CEX chromatography.

FIG. 170 depicts recovery versus AR reduction in the context of CEXpurification of adalimumab.

FIG. 171 depicts the WCX-10 profile of glycated load material and CEXeluate.

FIG. 172 depicts the WCX-10 profile of MGO modified load material andeluate from CEX column employing Toyo Pearl MX TRP 650M resin.

FIG. 173 depicts the change in lysine distribution during CEXchromatography, highlighting the effect of Tris concentration.

FIG. 174 depicts the effect of pH and conductivity on adalimumab ARreduction and recovery yield in the context of MM chromatography.

FIG. 175 depicts the AR reduction achieved with the correspondingprotein recovery in the context of MM chromatography.

FIG. 176 depicts the total adalimumab Protein concentration levels andAR levels during Flow Through and Wash.

FIG. 177 depicts the total mAb B Protein concentration levels and ARlevels during Flow Through and Wash in the context of MM chromatography.

FIG. 178 depicts the total mAb C Protein concentration levels and ARlevels during Flow Through and Wash in the context of MM chromatography.

FIG. 179 depicts the Cumulative % AR breakthrough of mAb C on differentMM resins.

FIG. 180 depicts the impact of pH-pI and conductivity on adalimumab ARreduction in the context of MM chromatography.

FIG. 181 depicts the impact of pH-pI and conductivity on mAb B ARreduction in the context of MM chromatography.

FIG. 182 depicts the impact and trend of pH-pI on mAb C AR reductionwith multiple resins in the context of MM chromatography.

FIG. 183 depicts the effect of pH and conductivity on AR reduction andYield in the context of MM chromatography.

FIG. 184 depicts AR reduction and protein recovery vs. pH in the contextof MM chromatography.

FIG. 185 depicts the effect of pH, conductivity and protein load amounton AR reduction and yield.

FIG. 186 depicts the effect of pH, conductivity and protein load amounton AR reduction and yield.

FIG. 187 depicts the effect of AEX adsorbent pKa for mAb B with severaldifferent AEX adsorbents, with different pKa values, run at with anacetate/Tris buffer at pH 9.1.

FIG. 188 is a schematic depiction of exemplary AR1 and AR2 present in acomposition comprising an exemplary antibody. Preparation-derived ARsand storage-derived ARs are depicted.

FIG. 189 depicts cumulative AR reduction as a function of yield forvarious formic acid concentrations.

FIG. 190 depicts an exemplary flow path for the production of a low ARcomposition.

FIG. 191 depicts an experimental scheme for a “ContinuousChromatography” process of producing a low AR composition.

FIG. 192 depicts the percent AR in each of the cycles of the continuousMM process.

FIG. 193 depicts a chromatogram wherein acidic and basic species areidentified in adalimumab and various fractions are delineated.

FIGS. 194A-B depict (A) the average arthritic scores and (B) growthrelated weight gain of mice administered low AR composition, AR1composition, Lys-1/2 composition, and control AR composition.

FIG. 195 depicts the average arthritic scores (area under the curve) ofmice administered low AR composition, AR1 composition, Lys-1/2composition, and control AR composition.

FIGS. 196A-B depict (A) the average trough serum drug levels and (B) theaverage trough serum ADA levels for mice administered low ARcomposition, AR1 composition, Lys-1/2 composition, and control ARcomposition.

FIG. 197 depicts the average PK and ADA profiles (area under the curve)for mice administered low AR composition, AR1 composition, Lys-1/2composition, and control AR composition.

FIG. 198 depicts complexed TNF levels (area under the curve) and showsthat the cumulative serum concentration values of adalimumab for miceadministered low AR composition, AR1 composition, Lys-1/2 composition,and control AR composition during the ten week treatment period washighest for the low AR and the control AR compositions and lowest forthe AR1 fraction.

FIG. 199 depicts the chondrocyte death, synovial proliferation,proteoglycan loss, cartilage destruction, and bone erosion of miceadministered low AR composition, AR1 composition, Lys-1/2 composition,and control AR composition.

FIGS. 200A-D illustrate the average drug levels for various tissues(paw, lymph node, spleen, skin, knee and serum) for mice administered(A) low AR composition; (B) control AR composition; (C) AR1 composition;and (D) Lys-1/2 composition.

FIGS. 201A-D illustrate the average ADA levels for various tissues (paw,lymph node, spleen, skin, knee and serum) for mice administered (A) lowAR composition; (B) control AR composition; (C) AR1 composition; and (D)Lys-1/2 composition.

FIGS. 202A-D show the results of a micro CT analysis of spines andfemurs obtained from TNF-Tg197 transgenic mice which were administeredplacebo, low AR composition, control (normal) AR composition, AR1composition, and Lys-1/2 composition. The graphs depict the effect ofthe administered compositions on (A) vertebra bone volume; (B) vertebratrabecular number; (C) vertebra trabecular thickness; and (D) vertebratrabecular space.

FIGS. 203A-D show the results of a micro CT analysis of spines andfemurs obtained from TNF-Tg197 transgenic mice which were administeredplacebo, low AR composition, control (normal) AR composition, AR1composition, and Lys-1/2 composition. The graphs depict the effect ofthe administered compositions on (A) vertebra bone loss; (B) vertebratrabecular number; (C) vertebra trabecular thickness; and (D) vertebratrabecular space.

FIGS. 204A-D show results of a micro CT analysis of spines and femursobtained from TNF-Tg197 transgenic mice which were administered placebo,low AR composition, control (normal) AR composition, AR1 composition,and Lys-1/2 composition. The graphs depict the effect of theadministered compositions on (A) trabecular bone volume/total volume atthe femoral metaphysis; (B) trabecular number at the femoral metaphysis;(C) trabecular thickness at the femoral metaphysis; and (D) trabecularseparation at the femoral metaphysis.

FIG. 205 depicts micro CT images of the spine from each of six groups ofmice administered the following compositions: naïve, vehicle (control),low AR composition (group 5), low host cell protein (HCP) composition(group 7), AR1 composition (containing only AR1 acidic variants) (group8), and Lys-1/2 composition (containing only Lys 1 and Lys 2 variants)(group 9).

FIG. 206 depicts micro CT images of the femur from each of six groups ofmice administered the following compositions: naïve, vehicle (control),low AR composition (group 5), low host cell protein (HCP) composition(group 7), AR1 composition (containing only AR1 acidic variants) (group8), and Lys-1/2 composition (containing only Lys 1 and Lys 2 variants)(group 9).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification and optimization ofupstream and downstream process technologies for protein production,e.g., production of antibodies or antigen-binding portions thereof,resulting in the production of protein compositions that comprise lowpercentages of acidic species (AR) and/or low levels of process-relatedimpurities (e.g., host cell proteins and media components).

As demonstrated herein, the compositions of the present inventionexhibit increased therapeutic efficacy when administered to a subject.For example, compositions comprising anti-TNFα antibodies, or antigenbinding portions thereof, comprising low AR are capable of increasedtherapeutic efficacy in the treatment and prevention of a disorder inwhich TNFα is detrimental, e.g., rheumatoid arthritis (RA), juvenileidiopathic arthritis (JIA), psoriasis, psoriatic arthritis, ankylosingspondylitis, Crohn's disease, and ulcerative colitis. Accordingly, theinstant invention provides compositions comprising proteins thatcomprise low AR and/or low levels of process-related impurities, andmethods for producing and using the same.

In one embodiment, the low AR compositions of the invention compriseabout 15% or less AR, 14% or less AR, 13% or less AR, 12% or less AR,11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or lessAR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5%or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9% or lessAR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR,1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1%or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% orless AR, 0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or lessAR, 0.1% or less AR, or 0.0% AR, and ranges within one or more of thepreceding. In one aspect of this embodiment, the low AR compositions ofthe invention comprise about 0.0% to about 10% AR, about 0.0% to about5% AR, about 0.0% to about 4% AR, about 0.0% to about 3% AR, about 0.0%to about 2% AR, about 3% to about 5% AR, about 5% to about 8% AR, orabout 8% to about 10% AR, or about 10% to about 15% AR, and rangeswithin one or more of the preceding. In one embodiment, the compositionof the invention is not a composition, e.g., an adalimumab composition,comprising 2.4% or 2.5% AR.

In another embodiment, the low AR composition comprises a first acidicspecies (AR1) and a second acidic species (AR2). In one aspect of thisembodiment, the low AR composition comprises about 0.1% or less AR1 andabout 3% or less AR2. In another aspect of this embodiment, the low ARcomposition comprises about 0.0% AR1 and about 1.4% or less AR2.

In another aspect of this embodiment, the low AR composition comprisesabout 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1 or less, 0.3% or less AR1 or less, 0.2% or less AR1 or less, 0.1% orless AR1, or 0.0% AR1, and ranges within one or more of the preceding.In one aspect of this embodiment, the low AR compositions of theinvention comprise about 0.0% to about 10% AR1, about 0.0% to about 5%AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% AR1, about 0.0%to about 2% AR1, about 3% to about 5% AR1, about 5% to about 8% AR1, orabout 8% to about 10% AR1, or about 10% to about 15% AR1, and rangeswithin one or more of the preceding. In one embodiment, the compositionof the invention is not a composition, e.g., an adalimumab composition,comprising 0.2% AR1.

In yet another aspect of this embodiment, the low AR compositioncomprises about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12%or less AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% orless AR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or lessAR2, 4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2,2% or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2,1.6% or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2,1.2% or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2,0.8% or less AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2,0.4% or less AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2,or 0.0% AR2, and ranges within one or more of the preceding. In oneaspect of this embodiment, the low AR compositions of the inventioncomprise about 0.0% to about 10% AR2, about 0.0% to about 5% AR2, about0.0% to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% to about 2%AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, or about 8% toabout 10% AR2, or about 10% to about 15% AR2, and ranges within one ormore of the preceding. In one embodiment, the composition of theinvention is not a composition, e.g., an adalimumab composition,comprising 2.2% AR2.

In another embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab, comprises about 1.4% or less AR. For example,in one aspect of this embodiment, the low AR composition, e.g., a low ARcomposition of adalimumab comprising about 1.4% or less AR comprisesabout 0.0% AR1 and about 1.4% or less AR2.

In one embodiment, the protein is an antibody or antigen binding portionthereof, such as adalimumab, or an antigen binding portion thereof.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, the terms “acidic species,” “acidic region,” and “AR,”refer to the variants of a protein, e.g., an antibody or antigen-bindingportion thereof, which are characterized by an overall acidic charge.For example, in monoclonal antibody (mAb) preparations, such acidicspecies can be detected by various methods, such as ion exchange, forexample, WCX-10 HPLC (a weak cation exchange chromatography), or IEF(isoelectric focusing). As depicted in FIG. 188, acidic species of anantibody may include charge variants, structure variants, and/orfragmentation variants. Exemplary charge variants include, but are notlimited to, deamidation variants, afucosylation variants, methylglyoxal(MGO) variants, glycation variants, and citric acid variants. Exemplarystructure variants include, but are not limited to, glycosylationvariants and acetonation variants. Exemplary fragmentation variantsinclude any truncated protein species from the target molecule due todissociation of peptide chain, enzymatic and/or chemical modifications,including, but not limited to, Fc and Fab fragments, fragments missing aFab, fragments missing a heavy chain variable domain, C-terminaltruncation variants, variants with excision of N-terminal Asp in thelight chain, and variants having N-terminal truncation of the lightchain. Other acidic species variants include variants containingunpaired disulfides, host cell proteins, and host nucleic acids,chromatographic materials, and media components.

In certain embodiments, a protein composition can comprise more than onetype of acidic species variant. For example, but not by way oflimitation, the total acidic species can be divided based onchromatographic retention time of the peaks appearing, for example, in aWCX-10 Weak Cation Exchange HPLC of the protein preparation. FIG. 163depicts a non-limiting example of such a division wherein the totalacidic species associated with the expression of adalimumab is dividedinto a first acidic species region (AR1) and a second acidic speciesregion (AR2).

As depicted schematically in FIG. 188, AR1 can comprise, for example,charge variants such as deamidation variants, MGO modified species,glycation variants, and citric acid variants, structural variants suchas glycosylation variants and acetonation variants, and/or fragmentationvariants. In another embodiment, AR2 can comprise, for example, chargevariants such as glycation variants and deamidation variants.

With respect, in particular, to adalimumab (and antibodies sharingcertain structural characteristics of adalimumab, e.g., one or more CDRand/or heavy and light chain variable regions of adalimumab), AR1 chargevariants can comprise, but are not limited to, deamidation variants,glycation variants, afucosylation variants, MGO (e.g., MGO variants atthe residues shown in Table 5, below) variants or citric acid variants.In one embodiment, deamidation variants result from deamidationoccurring at asparagine residues comprising Asn393 and Asn329 and atglutamine residues comprising Gln3 and Gln6. In another embodiment, theglycation variants result from glycation occurring at Lys98 and Lys151.AR1 structure variants can comprise, but are not limited to,glycosylation variants or acetonation variants.

AR1 fragmentation variants can comprise Fc and Fab fragments, fragmentsmissing a Fab, fragments missing a heavy chain variable domain,C-terminal truncation variants, variants with excision of N-terminal Aspin the light chain, and variants having N-terminal truncation of thelight chain.

AR2 charge variants can comprise, but are not limited to, deamidationvariants or glycation variants, wherein the deamidation variants canresult from deamidation occurring at asparagine residues comprisingAsn393 and Asn329 and at glutamine residues comprising Gln3 and Gln6,and the glycation variants can result from glycation occurring at Lys98and Lys151.

The term “acidic species” does not include process-related impurities.The term “process-related impurity,” as used herein, refers toimpurities that are present in a composition comprising a protein butare not derived from the protein itself. Process-related impuritiesinclude, but are not limited to, host cell proteins (HCPs), host cellnucleic acids, chromatographic materials, and media components. A “lowprocess-related impurity composition,” as used herein, refers to acomposition comprising reduced levels of process-related impurities ascompared to a composition wherein the impurities were not reduced. Forexample, a low process-related impurity composition may contain about10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less ofprocess-related impurities. In one embodiment, a low process-relatedimpurity composition is free of process-related impurities or issubstantially free of process-related impurities.

The acidic species may be the result of product preparation (referred toherein as “preparation-derived acidic species”), or the result ofstorage (referred to herein as “storage-derived acidic species”).Preparation-derived acidic species are acidic species that are formedduring the preparation (upstream and/or downstream processing) of theprotein, e.g., the antibody or antigen-binding portion thereof. Forexample, preparation-derived acidic species can be formed during cellculture (“cell culture-derived acidic species”). Storage-derived acidicspecies are acidic species that may or may not be present in thepopulation of proteins directly after preparation, but are formed orgenerated while the sample is being stored. The type and amount ofstorage-derived acidic species can vary based on the formulation of thesample. Formation of storage-derived acidic species can be partially orcompletely inhibited when the preparation is stored under particularconditions. For example, an aqueous formulation can be stored at aparticular temperature to partially or completely inhibit AR formation.For example, formation or storage-derived AR can be partially inhibitedin an aqueous formulation stored at between about 2° C. and 8° C., andcompletely inhibited when stored at −80° C. In addition, a low ARcomposition can be lyophilized or freeze-dried to partially orcompletely inhibit the formation of storage-derived AR.

The term “low acidic species composition,” or “low AR composition,” asused herein, refers to a composition comprising an antibody orantigen-binding portion thereof, wherein the composition contains lessthan about 15% acidic species. As used herein, the percent AR in the lowAR composition refers to the weight of the acidic species in a sample inrelation to the weight of the total antibodies contained in the sample.For example, the percent AR can be calculated using weak cation exchangechromatography such as WCX-10, as described in, for example, Example 1below.

In one embodiment, a low AR composition of the invention may compriseabout 15% or less AR, 14% or less AR, 13% or less AR, 12% or less AR,11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR, 7% or lessAR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5%or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9% or lessAR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR,1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1%or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% orless AR, 0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or lessAR, 0.1% or less AR, or 0.0% AR, and ranges within one or more of thepreceding. A low AR composition of the invention may also comprise about0.0% to about 10% AR, about 0.0% to about 5% AR, about 0.0% to about 4%AR, about 0.0% to about 3% AR, about 0.0% to about 2% AR, about 3% toabout 5% AR, about 5% to about 8% AR, or about 8% to about 10% AR, orabout 10% to about 15% AR, and ranges within one or more of thepreceding.

A low AR composition of the invention may comprise about 15% or lessAR1, 14% or less AR1, 13% or less AR1, 12% or less AR1, 11% or less AR1,10% or less AR1, 9% or less AR1, 8% or less AR1, 7% or less AR1, 6% orless AR1, 5% or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or lessAR1, 3% or less AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less AR1,1.8% or less AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less AR1,1.4% or less AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or less AR1,1% or less AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or less AR1,0.6% or less AR1, 0.5% or less AR1, 0.4% or less AR1, 0.3% or less AR1,0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1, and ranges within oneor more of the preceding. A low AR composition of the invention may alsocomprise about 0.0% to about 10% AR1, about 0.0% to about 5% AR1, about0.0% to about 4% AR1, about 0.0% to about 3% AR1, about 0.0% to about 2%AR1, about 3% to about 5% AR1, about 5% to about 8% AR1, or about 8% toabout 10% AR1, or about 10% to about 15% AR1, and ranges within one ormore of the preceding.

A low AR composition of the invention may also comprise about 15% orless AR2, 14% or less AR2, 13% or less AR2, 12% or less AR2, 11% or lessAR2, 10% or less AR2, 9% or less AR2, 8% or less AR2, 7% or less AR2, 6%or less AR2, 5% or less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% orless AR2, 3% or less AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or lessAR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or lessAR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or lessAR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or lessAR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% or lessAR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and ranges withinone or more of the preceding. A low AR composition of the invention mayalso comprise about 0.0% to about 10% AR2, about 0.0% to about 5% AR2,about 0.0% to about 4% AR2, about 0.0% to about 3% AR2, about 0.0% toabout 2% AR2, about 3% to about 5% AR2, about 5% to about 8% AR2, orabout 8% to about 10% AR2, or about 10% to about 15% AR2, and rangeswithin one or more of the preceding.

In one embodiment, a low AR composition comprises between about 0.0% andabout 3% AR1. In another embodiment, a low AR composition comprisesabout between about 0.0% and about 3% AR2. In still another embodiment,a low acidic species composition comprises about 3% or less AR2.

In another embodiment, the low AR composition comprises about 1.4% orless AR. For example, in one embodiment, the composition comprises about1.4% AR2 and about 0.0% AR1.

In one embodiment, a low AR composition of the invention may compriseabout 15% or less, 14% or less, 13% or less, 12% or less, 11% or less,10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% orless, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% orless, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% orless, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% orless, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, or 0.0% ofone or more of a deamidation variant, an afucosylation variant, an MGOvariant, a glycation variant, a citric acid variant, a glycosylationvariant, an acetonation variant, or a fragmentation variant, and rangeswithin one or more of the preceding. In one aspect of this embodiment, alow AR composition of the invention may also comprise about 0.0% toabout 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% toabout 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% toabout 8%, or about 8% to about 10%, or about 10% to about 15%, of one ormore of a deamidation variant, an afucosylation variant, an MGO variant,a glycation variant, a citric acid variant, a glycosylation variant, anacetonation variant, or a fragmentation variant, and ranges within oneor more of the preceding. For example, a low AR composition of theinvention may comprise less than 15% of a deamidation variant, whileeach of the other acidic variants, alone or in combination, are at apercentage that is greater than 15%.

The term “non-low acidic species composition,” as used herein, refers toa composition comprising an antibody or antigen-binding portion thereof,which contains more than about 16% acidic species. For example, anon-low acidic species composition may contain about 16% or more, 17% ormore, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more,23% or more, 24% or more, or 25% or more acidic species. In oneembodiment, a non-low acidic species composition can comprise about 16%or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% ormore, 22% or more, 23% or more, 24% or more, or 25% or more of AR1. Inanother embodiment, a non-low acidic species composition can compriseabout 16% or more, 17% or more, 18% or more, 19% or more, 20% or more,21% or more, 22% or more, 23% or more, 24% or more, or 25% or more ofAR2, and ranges within one or more of the preceding.

In one embodiment, a low AR composition has improved biological andfunctional properties, including increased efficacy in the treatment orprevention of a disorder in a subject, e.g., a disorder in which TNFαactivity is detrimental, as compared to a non-low acidic speciescomposition. In one embodiment, the low AR composition comprises ananti-TNFα antibody, or antigen-binding portion thereof, such asadalimumab or a fragment thereof. For example, in one embodiment, a lowAR composition comprising an antibody, or antigen-binding portionthereof, exhibits increased cartilage penetration, decreased boneerosion, and/or reduced cartilage destruction, as compared to a non-lowacidic species composition comprising the same antibody or antigenbinding portion thereof, when administered to a subject suffering from adisorder in which TNFα activity is detrimental.

As used herein, the term “increased cartilage penetration” refers toincreased penetration of cartilage in vivo by a low AR composition ascompared to a non-low AR composition comprising the same antibody orantigen binding portion thereof.

As used herein, the term “reduced cartilage destruction” refers tomeasurable decrease in destruction of cartilage tissue in vivo by a lowAR composition as compared to a non-low AR composition comprising thesame antibody or antigen binding portion thereof. As used herein, theterm “decreased bone erosion” refers to measurable decrease, in vivo, ofthe erosion of bone tissue by a low AR composition as compared to anon-low acidic species composition comprising the same antibody orantigen binding portion thereof. For example, an in vivo model of adisease or disorder in which TNFα activity is detrimental, e.g., a mousemodel of arthritis, can be used to measure cartilage penetration, boneerosion, and/or cartilage destruction by a composition comprising ananti-TNFα antibody or antigen binding portion thereof. One non-limitingexample of an art-recognized mouse model of arthritis is the human TNFtransgenic 197 mouse model of arthritis (TNF-Tg197) (see Keffer, J. etal., EMBO J (1991) 10:4025-4031, the contents of which are expresslyincorporated herein by reference, for further description of theTNF-Tg197 model of arthritis).

In another embodiment, a low AR composition comprising an antibody, orantigen-binding portion thereof, exhibits increased protection againstthe development of arthritis, as measured by arthritic scores, and/orhistopathology scores as compared to a non-low acidic speciescomposition when administered to an animal model of arthritis, e.g., theTNF-Tg197 model of arthritis. As used herein, “arthritic scores” referto signs and symptoms of arthritis in an animal model of arthritis. Asused herein, “histopathology scores” refer to radiologic damageinvolving cartilage and bone as well as local inflammation.

In another embodiment, a low AR composition comprising an antibody, orantigen-binding portion thereof, exhibits reduced synovialproliferation, reduced cell infiltration, reduced chondrocyte death,and/or reduced proteoglycan loss as compared to a non-low acidic speciescomposition. In another embodiment, a low AR composition comprising ananti-TNFα antibody, or antigen-binding portion thereof, exhibitsincreased TNFα affinity as compared to a non-low acidic speciescomposition.

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, or synovial fluid of the subject), which can bedetected, for example, using an anti-TNFα antibody as described above.There are numerous examples of disorders in which TNFα activity isdetrimental. In one embodiment, the disorder in which TNFα activity isdetrimental is an autoimmune disorder. In one embodiment, the autoimmunedisorder is selected from the group consisting of rheumatoid arthritis,juvenile idiopathic arthritis, rheumatoid spondylitis, ankylosingspondylitis, psoriasis, osteoarthritis, gouty arthritis, an allergy,multiple sclerosis, psoriatic arthritis, autoimmune diabetes, autoimmuneuveitis, nephrotic syndrome, juvenile rheumatoid arthritis, Crohn'sdisease, ulcerative colitis, active axial spondyloarthritis (activeaxSpA) and non-radiographic axial spondyloarthritis (nr-axSpA).Disorders in which TNFα activity is detrimental are set forth in U.S.Pat. No. 6,090,382 and also in the “Highlights of PrescribingInformation” for HUMIRA® (adalimumab) Injection (Revised January 2008)the contents of which are hereby incorporated herein by reference. Theuse of TNFα antibodies and antibody portions obtained using methods ofthe invention for the treatment of specific disorders is discussed infurther detail below.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., in the case of adalimumab,hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment comprising the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentcomprising the VH and CH1 domains; (iv) a Fv fragment comprising the VLand VH domains of a single arm of an antibody, (v) a dAb fragment (Wardet al., (1989) Nature 341:544-546, the entire teaching of which isincorporated herein by reference), which comprises a VH domain; and (vi)an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see, e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883, the entire teachings of which are incorporated herein byreference). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody or antigen-binding portion thereof may be part of alarger immunoadhesion molecule, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101, the entire teaching of which is incorporated herein byreference) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058,the entire teaching of which is incorporated herein by reference).Antibody portions, such as Fab and F(ab′)2 fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.In one aspect, the antigen binding portions are complete domains orpairs of complete domains.

The terms “Kabat numbering” “Kabat definitions” and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, the entire teachings ofwhich are incorporated herein by reference). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hTNFα is substantially free ofantibodies that specifically bind antigens other than hTNFα). Anisolated antibody that specifically binds hTNFα may bind TNFα moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals. A suitable anti-TNFαantibody is adalimumab.

As used herein, the term “adalimumab,” also known by its trade nameHUMIRA® (AbbVie) refers to a human IgG₁ antibody that binds human tumornecrosis factor α (TNFα). In general, the heavy chain constant domain 2(CH2) of the adalimumab IgG-Fc region is glycosylated through covalentattachment of oligosaccharide at asparagine 297 (Asn-297). The lightchain variable region of adalimumab is provided herein as SEQ ID NO:1,and the heavy chain variable region of adalimumab is provided herein asSEQ ID NO:2. Adalimumab comprises a light chain variable regioncomprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID NO:5, and a CDR3 ofSEQ ID NO:3. Adalimumab comprises a heavy chain variable regioncomprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:6 and CDR3 of SEQID NO:4. The nucleic acid sequence of the light chain variable region isset forth in SEQ ID NO:9. The nucleic acid sequence of the heavy chainvariable region is set forth in SEQ ID NO:10. The full length amino acidsequence of the light chain is set forth as SEQ ID NO:11 and the fulllength amino acid sequence of the heavy chain is set forth as SEQ IDNO:12. Adalimumab is described in U.S. Pat. Nos. 6,090,382; 6,258,562;6,509,015; 7,223,394; 7,541,031; 7,588,761; 7,863,426; 7,919,264;8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998; 8,093,045;8,187,836; 8,372,400; 8,034,906; 8,436,149; 8,231,876; 8,414,894;8,372,401, the entire contents of each which are expressly incorporatedherein by reference in their entireties. Adalimumab is also described inthe “Highlights of Prescribing Information” for HUMIRA® (adalimumab)Injection (Revised January 2008) the contents of which are herebyincorporated herein by reference.

In one embodiment, adalimumab dissociates from human TNFα with a Kd of1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10-M orless. In another embodiment, adalimumab dissociates from human TNFα witha K_(off) of 5×10⁴ s⁻¹ or less, or with a K_(off) of 1×10⁻⁴ s⁻¹ or less.In still another embodiment, adalimumab neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10⁻⁸ Mor less, an IC50 of 1×10⁻⁹ M or less or an IC50 of 1×10⁻¹⁰ M or less.

In general, the heavy chain constant domain 2 (CH2) of the adalimumabIgG-Fc region is glycosylated through covalent attachment ofoligosaccharide at asparagine 297 (Asn-297). Analysis of adalimumab hasshown that it has three main basic variants (i.e., Lys 0, Lys 1, and Lys2), referred to herein as “lysine variant species.” As used herein, theterm “lysine variant species” refers to an antibody, or antigen-bindingportion thereof, comprising heavy chains with either zero, one or twoC-terminal lysines. For example, the “Lys 0” variant comprises anantibody, or antigen-binding portion thereof, with heavy chains that donot comprise a C-terminal lysine. The “Lys 1” variant comprises anantibody, or antigen-binding portion thereof, with one heavy chain thatcomprises a C-terminal lysine. The “Lys 2” variant comprises an antibodywith both heavy chains comprising a C-terminal lysine. Lysine variantscan be detected, for example, by weak cation exchange chromatography(such as WCX-10) of the expression product of a host cell expressing theantibody, or antigen-binding portion thereof. For example, but not byway of limitation, FIGS. 163 and 193 depict WCX-10 analysis ofadalimumab wherein the three lysine variants, as well as the two acidicspecies regions, are resolved from each other.

A composition of the invention may comprise more than one lysine variantspecies of an antibody, or antigen-binding portion thereof. For example,in one embodiment, the composition may comprise a Lys 2 variant of anantibody, or antigen-binding portion thereof. The composition maycomprise a Lys 1 variant of an antibody, or antigen-binding portionthereof. The composition may comprise a Lys 0 variant of an antibody, orantigen-binding portion thereof. In another embodiment, the compositionmay comprise both Lys 1 and Lys 2 or Lys 1 and Lys 0 or Lys 2 and Lys 0variants of an antibody, or antigen-binding portion thereof. In anotherembodiment, the composition may comprise all three lysine variantspecies, i.e., Lys 0, Lys 1 and Lys 2, of an antibody, orantigen-binding portion thereof.

As used herein, the term “upstream process technology,” in the contextof protein, e.g., antibody, preparation, refers to activities involvingthe production and collection of proteins (e.g. antibodies) from cells(e.g., during cell culture of a protein of interest). As used herein,the term “cell culture” refers to methods for generating and maintaininga population of host cells capable of producing a recombinant protein ofinterest, as well as the methods and techniques for optimizing theproduction and collection of the protein of interest. For example, oncean expression vector has been incorporated into an appropriate host, thehost can be maintained under conditions suitable for expression of therelevant nucleotide coding sequences, and the collection andpurification of the desired recombinant protein.

When using the cell culture techniques of the instant invention, theprotein of interest can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. In embodiments where theprotein of interest is produced intracellularly, the particulate debris,either host cells or lysed cells (e.g., resulting from homogenization)can be removed by a variety of means, including but not limited to,centrifugation or ultrafiltration. Where the protein of interest issecreted into the medium, supernatants from such expression systems canbe first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit.

As used herein, the term “downstream process technology” refers to oneor more techniques used after the upstream process technologies topurify the protein, e.g., antibody, of interest. For example, downstreamprocess technology includes purification of the protein product, using,for example, affinity chromatography, including Protein A affinitychromatography, ion exchange chromatography, such as anion or cationexchange chromatography, hydrophobic interaction chromatography, ordisplacement chromatography.

The phrase “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3), e.g., those that bind hTNFα, includes a nucleic acid molecule inwhich the nucleotide sequences encoding the antibody or antibody portionare free of other nucleotide sequences encoding antibodies or antibodyportions that bind antigens other than hTNFα, which other sequences maynaturally flank the nucleic acid in human genomic DNA. Thus, e.g., anisolated nucleic acid of the invention encoding a VH region of ananti-TNFα antibody contains no other sequences encoding other VH regionsthat bind antigens other than, for example, hTNFα. The phrase “isolatednucleic acid molecule” is also intended to include sequences encodingbivalent, bispecific antibodies, such as diabodies in which VH and VLregions contain no other sequences other than the sequences of thediabody.

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “recombinant protein” refers to a proteinproduced as the result of the transcription and translation of a genecarried on a recombinant expression vector that has been introduced intoa host cell. In certain embodiments the recombinant protein is anantibody, e.g., a chimeric, humanized, or fully human antibody. Incertain embodiments the recombinant protein is an antibody of an isotypeselected from group consisting of: IgG (e.g., IgG1, IgG2, IgG3, IgG4),IgM, IgA1, IgA2, IgD, or IgE. In certain embodiments the antibodymolecule is a full-length antibody (e.g., an IgG1 or IgG4immunoglobulin) or alternatively the antibody can be a fragment (e.g.,an Fc fragment or a Fab fragment).

The phrase “clarified harvest” refers to a liquid material containing aprotein of interest, for example, an antibody of interest such as amonoclonal antibody of interest, that has been extracted from cellculture, for example, a fermentation bioreactor, after undergoingcentrifugation to remove large solid particles and subsequent filtrationto remove finer solid particles and impurities from the material.

The term “preparative scale,” as used herein, refers to a scale ofpurification operation that can be readily scaled-up and implemented atlarge scale manufacturing while still providing desired separation. Forinstance, one skilled in the field may develop a process using, e.g., a0.5 cm (i.d.)×20 cm (L) column in the lab, and transfer it to largescale production using, e.g., a 30 cm (i.d.)×20 cm (L) column packedwith the same resin and operated with the same set of buffers, samelinear flow rates (or residence times) and buffer volumes. Inpreparative scale separation, column bed height is typically ≦about 30cm and column pressure drop ≦about 5 bar.

II. Low Acidic Species Compositions of the Invention

The present invention provides low AR compositions comprising a protein,e.g., an antibody, or antigen-binding portion thereof, such asadalimumab, where the composition comprises about 15% or less AR, 14% orless AR, 13% or less AR, 12% or less AR, 11% or less AR, 10% or less AR,9% or less AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or lessAR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5%or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% orless AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or lessAR, 1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4%or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0%AR, and ranges within one or more of the preceding. A low AR compositionof the invention may also comprise about 0.0% to about 10% AR, about0.0% to about 5% AR, about 0.0% to about 4% AR, about 0.0% to about 3%AR, about 0.0% to about 2% AR, about 3% to about 5% AR, about 5% toabout 8% AR, or about 8% to about 10% AR, or about 10% to about 15% AR,and ranges within one or more of the preceding.

In one embodiment, a low AR composition of the invention may compriseabout 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% or lessAR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or less AR1,7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1, 4% orless AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2% or lessAR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or lessAR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or lessAR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% or lessAR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or lessAR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1,and ranges within one or more of the preceding. A low AR composition ofthe invention may also comprise about 0.0% to about 10% AR1, about 0.0%to about 5% AR1, about 0.0% to about 4% AR1, about 0.0% to about 3% AR1,about 0.0% to about 2% AR1, about 3% to about 5% AR1, about 5% to about8% AR1, or about 8% to about 10% AR1, or about 10% to about 15% AR1, andranges within one or more of the preceding.

In another embodiment, a low AR composition of the invention may alsocomprise about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12% orless AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% or lessAR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2,4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2%or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6%or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2%or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% orless AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% orless AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0%AR2, and ranges within one or more of the preceding. A low ARcomposition of the invention may also comprise about 0.0% to about 10%AR2, about 0.0% to about 5% AR2, about 0.0% to about 4% AR2, about 0.0%to about 3% AR2, about 0.0% to about 2% AR2, about 3% to about 5% AR2,about 5% to about 8% AR2, or about 8% to about 10% AR2, or about 10% toabout 15% AR2, and ranges within one or more of the preceding.

As demonstrated herein, these low AR compositions have improvedbiological properties (see Example 13). For example, the low ARcompositions of the invention are characterized by increased cartilagetissue penetration, reduced cartilage destruction, reduced synovialproliferation, reduced bone erosion, increased protection against thedevelopment of arthritic scores and/or histopathology scores, reducedcell infiltration, reduced proteoglycan loss, reduced chondrocyte death,and/or increased TNF affinity, as compared to non-low acidic speciescompositions. In addition, the compositions of the present inventionexhibit increased therapeutic efficacy when administered to a subject.

In one embodiment, the protein in the low AR composition of theinvention is an antibody or antigen binding portion thereof. Forexample, the antibody, or antigen binding portion thereof may be ananti-TNFα antibody, or antigen binding portion thereof, such asadalimumab, or an antigen binding portion thereof. In one aspect of thisembodiment, the antibody, or antigen binding portion thereof, cancomprise a light chain variable region comprising the sequence set forthas SEQ ID NO:1, and a heavy chain variable region comprising thesequence set forth as SEQ ID NO:2. In another aspect of this embodiment,the antibody can comprise a light chain variable region comprising aCDR1 having the sequence set forth as SEQ ID NO:7, a CDR2 having thesequence set forth as SEQ ID NO:5, and a CDR3 having the sequence setforth as SEQ ID NO:3. In another aspect of this embodiment, the antibodycan comprise a heavy chain variable region comprising a CDR1 having thesequence set forth as SEQ ID NO:8, a CDR2 having the sequence set forthas SEQ ID NO:6 and a CDR3 having the sequence set forth as SEQ ID NO:4.

The antibody, or antigen binding portion thereof, used in the low ARcompositions of the invention, may be a human, humanized, or chimericantibody.

The antibodies that can be used in the low AR compositions of thepresent disclosure can be generated by a variety of techniques,including immunization of an animal with the antigen of interestfollowed by conventional monoclonal antibody methodologies e.g., thestandard somatic cell hybridization technique of Kohler and Milstein(1975) Nature 256: 495. Somatic cell hybridization procedures can beused. In principle, other techniques for producing monoclonal antibodycan be employed as well, including viral or oncogenic transformation ofB lymphocytes.

One exemplary animal system for preparing hybridomas is the murinesystem. Hybridoma production is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

An antibody used in the low AR compositions of the invention can be ahuman, a chimeric, or a humanized antibody. Chimeric or humanizedantibodies used in the low AR compositions of the invention can beprepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

In one non-limiting embodiment, the antibodies to be used in the low ARcompositions of the invention are human monoclonal antibodies. Suchhuman monoclonal antibodies can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KMMouse® (Medarex, Inc.), and XenoMouse® (Amgen). The antibodies, orantigen-binding portions thereof, used in the low AR compositions of theinvention can also be produced using the methods described in U.S. Pat.No. 6,090,382, the entire contents of which is expressly incorporatedherein by reference.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseantibodies of the disclosure. For example, mice carrying both a humanheavy chain transchromosome and a human light chain transchromosome,referred to as “TC mice” can be used; such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (e.g., Kuroiwa et al. (2002) NatureBiotechnology 20:889-894 and PCT application No. WO 2002/092812) and canbe used to raise antibodies of this disclosure.

Recombinant human antibodies to be used in the low AR compositions ofthe invention can be isolated by screening of a recombinantcombinatorial antibody library, e.g., a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612, the entireteachings of which are incorporated herein), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, e.g., Ladner et al. U.S.Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Doweret al. PCT Publication No. WO 91/17271; Winter et al. PCT PublicationNo. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679;Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCTPublication No. WO 92/01047; Garrard et al. PCT Publication No. WO92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibody Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982; the entire teachings of whichare incorporated herein.

Human monoclonal antibodies to be used in the low AR compositions of theinvention can also be prepared using SCID mice into which human immunecells have been reconstituted such that a human antibody response can begenerated upon immunization. Such mice are described in, for example,U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

In certain embodiments, the human antibodies to be used in the low ARcompositions of the invention are anti-TNFα antibodies and antibodyportions thereof, anti-TNFα-related antibodies and antibody portions,and human antibodies and antibody portions with equivalent properties toanti-TNFα antibodies, such as high affinity binding to hTNFα with lowdissociation kinetics and high neutralizing capacity. In one aspect, theinvention provides low AR compositions containing an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhTNFα with a Kd of about 1×10⁻⁸ M or less and a Koff rate constant of1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance. Inspecific non-limiting embodiments, an anti-TNFα antibody to be used inthe low AR compositions of the invention competitively inhibits bindingof adalimumab to TNFα under physiological conditions. In one embodiment,the low AR compositions of the invention comprise adalimumab, or anantigen binding fragment thereof.

Antibodies or fragments thereof to be used in the low AR compositions ofthe invention can be altered wherein the constant region of the antibodyis modified to reduce at least one constant region-mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor, the immunoglobulin constant region segment of the antibodycan be mutated at particular regions necessary for Fc receptor (FcR)interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med.173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662, theentire teachings of which are incorporated herein). Reduction in FcRbinding ability of the antibody may also reduce other effector functionswhich rely on FcR interactions, such as opsonization and phagocytosisand antigen-dependent cellular cytotoxicity.

III. Preparation of Low AR Compositions Using Upstream ProcessTechnologies

The low AR compositions comprising a protein, e.g., an antibody, orantigen binding portion thereof, such as adalimumab, of the inventioncan be produced by modulating conditions during upstream proteinproduction, such as cell culture. In one embodiment, the methods of theinvention comprise lowering the amount of acidic species variants orprocess-related impurities expressed by host cells producing a proteinof interest including an antibody or antigen-binding portion thereofduring an upstream process technology (e.g., during cell culture).

The upstream process technologies may be used alone or in combinationwith the downstream process technologies described in Section IV, below,and as described in Example 10.

In one embodiment, one or more of the upstream process technologiesdescribed herein produce a low AR composition comprising an antibody, orantigen binding portion thereof, which comprises 15% or less AR, 14% orless AR, 13% or less AR, 12% or less AR, 11% or less AR, 10% or less AR,9% or less AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or lessAR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5%or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% orless AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or lessAR, 1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4%AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0% AR, andranges within one or more of the preceding. In one aspect of thisembodiment, the low AR composition of the invention comprises about 0.0%to about 10% AR, about 0.0% to about 5% AR, about 0.0% to about 4% AR,about 0.0% to about 3% AR, about 0.0% to about 2% AR, about 3% to about5% AR, about 5% to about 8% AR, or about 8% to about 10% AR, or about10% to about 15% AR, and ranges within one or more of the preceding.

In another embodiment, one or more of the upstream process technologiesdescribed herein produce a low AR composition comprising an antibody, orantigen binding portion thereof, which comprises 15% or less AR1, 14% orless AR1, 13% or less AR1, 12% or less AR1, 11% or less AR1, 10% or lessAR1, 9% or less AR1, 8% or less AR1, 7% or less AR1, 6% or less AR1, 5%or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3% orless AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less AR1, 1.8% orless AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less AR1, 1.4% orless AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or less AR1, 1% orless AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or less AR1, 0.6% orless AR1, 0.5% or less AR1, 0.4% or less AR1, 0.3% or less AR1, 0.2% orless AR1, 0.1% or less AR1, or 0.0% AR1, and ranges within one or moreof the preceding. In one aspect of this embodiment, the low ARcomposition of the invention comprises about 0.0% to about 10% AR1,about 0.0% to about 5% AR1, about 0.0% to about 4% AR1, about 0.0% toabout 3% AR1, about 0.0% to about 2% AR1, about 3% to about 5% AR1,about 5% to about 8% AR1, or about 8% to about 10% AR1, or about 10% toabout 15% AR1, and ranges within one or more of the preceding.

In still another embodiment, one or more of the upstream processtechnologies described herein produce a low AR composition comprising anantibody, or antigen binding portion thereof, which comprises 15% orless AR2, 14% or less AR2, 13% or less AR2, 12% or less AR2, 11% or lessAR2, 10% or less AR2, 9% or less AR2, 8% or less AR2, 7% or less AR2, 6%or less AR2, 5% or less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% orless AR2, 3% or less AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or lessAR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or lessAR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or lessAR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or lessAR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% or lessAR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and ranges withinone or more of the preceding. In one aspect of this embodiment, the lowAR composition of the invention comprises about 0.0% to about 10% AR2,about 0.0% to about 5% AR2, about 0.0% to about 4% AR2, about 0.0% toabout 3% AR2, about 0.0% to about 2% AR2, about 3% to about 5% AR2,about 5% to about 8% AR2, or about 8% to about 10% AR2, or about 10% toabout 15% AR2, and ranges within one or more of the preceding.

Some embodiments of the invention comprise culturing host cells toexpress a protein of interest under conditions that limit the amount ofacidic species that are expressed by the cells. Some embodiments of theinvention comprise culturing host cells under conditions that limit theconversion of the product to acidic species variants.

The cell culture conditions can be modified as compared to conditionsduring production of a non-low acidic species composition comprising thesame protein. In one embodiment, the low acidic species composition isproduced by culturing cells expressing the antibody, or antigen bindingportion thereof, in a cell culture media comprising an increasedconcentration of one or more amino acids. In another embodiment, the lowacidic species composition is produced by culturing cells expressing theantibody, or antigen binding portion thereof, in a cell culture mediacomprising an increased concentration of calcium (e.g., as calciumchloride dihydrate). In still another embodiment, the low acidic speciescomposition is produced by culturing cells expressing the antibody, orantigen binding portion thereof, in a cell culture media comprising anincreased concentration of niacinamide. In certain embodiments, themethods described herein comprise culturing cells in media supplementedwith one or more amino acids, calcium (e.g., as calcium chloridedihydrate) and/or niacinamide, and combinations thereof.

In certain embodiments, the low acidic species composition is producedby culturing host cells in a culture wherein process parameters, such aspH or dissolved oxygen (DO), are modulated, e.g., lowered to decreasethe amount of acidic species produced by the host cells and/or reducethe conversion of the product to the acidic species variants.

Furthermore, a continuous or perfusion technology can utilized to obtainlow AR. In certain embodiments, reduction of acidic species is obtainedby modulating the medium exchange rate during cell culture.

In another embodiment, one or more of the above supplements andmodifications can be combined and used during cell culture of oneprotein, e.g., antibody, composition.

To express an antibody or antigen-binding fragment thereof to be used inthe low AR compositions of the invention, DNAs encoding the protein,such as DNAs encoding partial or full-length light and heavy chains inthe case of antibodies, are inserted into one or more expression vectorsuch that the genes are operatively linked to transcriptional andtranslational control sequences. (See, e.g., U.S. Pat. No. 6,090,382,the entire teaching of which is incorporated herein by reference.) Inthis context, the term “operatively linked” is intended to mean that agene encoding the protein of interest is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the gene. The expression vector and expression controlsequences are chosen to be compatible with the expression host cellused. In certain embodiments, the protein of interest will comprisingmultiple polypeptides, such as the heavy and light chains of anantibody. Thus, in certain embodiments, genes encoding multiplepolypeptides, such as antibody light chain genes and antibody heavychain genes, can be inserted into a separate vector or, more typically,the genes are inserted into the same expression vector. Genes areinserted into expression vectors by standard methods (e.g., ligation ofcomplementary restriction sites on the gene fragment and vector, orblunt end ligation if no restriction sites are present). Prior toinsertion of the gene or genes, the expression vector may already carryadditional polypeptide sequences, such as, but not limited to, antibodyconstant region sequences. For example, one approach to converting theanti-TNFα antibody or anti-TNFα antibody-related VH and VL sequences tofull-length antibody genes is to insert them into expression vectorsalready encoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to the CHsegment(s) within the vector and the VL segment is operatively linked tothe CL segment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the protein from a host cell. The gene can becloned into the vector such that the signal peptide is linked in-frameto the amino terminus of the gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to protein coding genes, a recombinant expression vector cancarry one or more regulatory sequence that controls the expression ofthe protein coding genes in a host cell. The term “regulatory sequence”is intended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the protein coding genes. Such regulatory sequencesare described, e.g., in Goeddel; Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990), the entireteaching of which is incorporated herein by reference. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Suitable regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. For further description of viralregulatory elements, and sequences thereof, see, e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al., the entire teachings of whichare incorporated herein by reference.

A recombinant expression vector may also carry one or more additionalsequences, such as a sequence that regulates replication of the vectorin host cells (e.g., origins of replication) and/or a selectable markergene. The selectable marker gene facilitates selection of host cellsinto which the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al., the entireteachings of which are incorporated herein by reference). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr− host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

An antibody, or antibody portion, to be used in the low AR compositionsof the invention can be prepared by recombinant expression ofimmunoglobulin light and heavy chain genes in a host cell. To express anantibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and secreted intothe medium in which the host cells are cultured, from which medium theantibodies can be recovered. Standard recombinant DNA methodologies areused to obtain antibody heavy and light chain genes, incorporate thesegenes into recombinant expression vectors and introduce the vectors intohost cells, such as those described in Sambrook, Fritsch and Maniatis(eds), Molecular Cloning; A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 4,816,397 & 6,914,128, the entire teachings of which areincorporated herein.

For expression of protein, for example, the light and heavy chains of anantibody, the expression vector(s) encoding the protein is (are)transfected into a host cell by standard techniques. The various formsof the term “transfection” are intended to encompass a wide variety oftechniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is theoretically possible to express the proteins of theinvention in either prokaryotic or eukaryotic host cells, expression ofantibodies in eukaryotic cells, such as mammalian host cells, issuitable because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active protein. Prokaryoticexpression of protein genes has been reported to be ineffective forproduction of high yields of active protein (Boss and Wood (1985)Immunology Today 6:12-13, the entire teaching of which is incorporatedherein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated proteins, forexample, glycosylated antibodies, are derived from multicellularorganisms. Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori have beenidentified. A variety of viral strains for transfection are publiclyavailable, e.g., the L-1 variant of Autographa californica NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Mammalian cells can be used for expression and production of therecombinant protein used in the low AR compositions of the invention,however other eukaryotic cell types can also be employed in the contextof the instant invention. See, e.g., Winnacker, From Genes to Clones,VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells forexpressing recombinant proteins according to the invention includeChinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, describedin Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp (1982) Mol.Biol. 159:601-621, the entire teachings of which are incorporated hereinby reference), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding protein genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or secretion of the antibody into theculture medium in which the host cells are grown. Other examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2), the entire teachings of which areincorporated herein by reference.

Host cells are transformed with the above-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce a protein may be cultured in a variety ofmedia. Commercially available media such as Ham's F10™ (Sigma), MinimalEssential Medium™ (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium™ (DMEM), (Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells, the entire teachings of which areincorporated herein by reference. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asgentamycin drug), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

Host cells can also be used to produce portions of intact proteins, forexample, antibodies, including Fab fragments or scFv molecules. It isunderstood that variations on the above procedure are within the scopeof the present invention. For example, in certain embodiments it may bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an antibody. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to an antigen. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the target antibody,depending on the specificity of the antibody of the invention, bycrosslinking an antibody of the invention to a second antibody bystandard chemical crosslinking methods.

In a suitable system for recombinant expression of a protein, forexample, an antibody, or antigen-binding portion thereof, a recombinantexpression vector encoding the protein, for example, both an antibodyheavy chain and an antibody light chain, is introduced into dhfr-CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the protein gene(s) are each operatively linked toCMV enhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the gene(s). The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the protein, for example, theantibody heavy and light chains, and intact protein, for example, anantibody, is recovered from the culture medium. Standard molecularbiology techniques are used to prepare the recombinant expressionvector, transfect the host cells, select for transformants, culture thehost cells and recover the protein from the culture medium.

When using recombinant techniques, the protein, for example, antibodiesor antigen binding fragments thereof, can be produced intracellularly,in the periplasmic space, or directly secreted into the medium. In oneaspect, if the protein is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed cells (e.g., resultingfrom homogenization), can be removed, e.g., by centrifugation orultrafiltration. Where the protein is secreted into the medium,supernatants from such expression systems can be first concentratedusing a commercially available protein concentration filter, e.g., anAmicon™ or Millipore Pellicon™ ultrafiltration unit.

Some antibodies can be secreted directly from the cell into thesurrounding growth media; others are made intracellularly. Forantibodies made intracellularly, the first step of a purificationprocess typically involves: lysis of the cell, which can be done by avariety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by differential centrifugation or by filtration.Where the antibody is secreted, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

Adjusting Amino Acid Concentration to Modulate Acidic Species (AR)

In certain embodiments, the amount of one or more amino acids in themedia is modulated (e.g., increased or decreased) in order to produce alow acidic species composition of the invention (see the ExamplesSection, below). Such increases or decreases in the amount of the one ormore amino acids can be of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, andranges within one or more of the preceding, of the original amount usedduring cell culture where a non-low acidic species composition of thesame protein is produced.

In certain embodiments, a cell culture media will include one or more ofthe amino acids, or other compositions described herein as loweringacidic species. Accordingly, the amount of the amino acid, or othercomposition, that is supplemented may be adjusted to account for theamount present in the media prior to supplementation.

In certain embodiments, the cell culture media is supplemented with oneor more amino acids in an amount of between about 0.025 and 20 g/L, orbetween about 0.05 and 15 g/L, or between about 0.1 and 14 g/L, orbetween about 0.2 and 13 g/L, or between about 0.25 and 12 g/L, orbetween about 0.5 and 11 g/L, or between about 1 and 10 g/L, or betweenabout 1.5 and 9.5 g/L, or between about 2 and 9 g/L, or between about2.5 and 8.5 g/L, or between about 3 and 8 g/L, or between about 3.5 and7.5 g/L, or between about 4 and 7 g/L, or between about 4.5 and 6.5 g/L,or between about 5 and 6 g/L. In certain embodiments, the cell culturemedia is supplemented with one or more amino acids in an amount of about0.25 g/L, or about 0.5 g/L, or about 1 g/L, or about 2 g/L, or about 4g/L, or about 8 g/L.

In certain embodiments, the cell culture media is supplemented with oneor more amino acids in an amount effective to produce a low ARcomposition comprising about 15% or less AR, 14% or less AR, 13% or lessAR, 12% or less AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% orless AR, 7% or less AR, 6% or less AR, 5% or less AR, 4.5% or less AR,4% or less AR, 3.5% or less AR, 3% or less AR, 2.5% or less AR, 2% orless AR, 1.9% or less AR, 1.8% or less AR, 1.7% or less AR, 1.6% or lessAR, 1.5% or less AR, 1.4% or less AR, 1.3% or less AR, 1.2% or less AR,1.1% or less AR, 1% or less AR, 0.9% or less AR, 0.8% or less AR, 0.7%or less AR, 0.6% or less AR, 0.5% or less AR, 0.4% or less AR, 0.3% orless AR, 0.2% or less AR, 0.1% or less AR, or 0.0% AR, and ranges withinone or more of the preceding.

In another embodiment, the cell culture media is supplemented with oneor more amino acids in an amount effective to produce a low ARcomposition comprising about 15% or less AR1, 14% or less AR1, 13% orless AR1, 12% or less AR1, 11% or less AR1, 10% or less AR1, 9% or lessAR1, 8% or less AR1, 7% or less AR1, 6% or less AR1, 5% or less AR1,4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3% or less AR1, 2.5%or less AR1, 2% or less AR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% orless AR1, 1.6% or less AR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% orless AR1, 1.2% or less AR1, 1.1% or less AR1, 1% or less AR1, 0.9% orless AR1, 0.8% or less AR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% orless AR1, 0.4% or less AR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% orless AR1, or 0.0% AR1, and ranges within one or more of the preceding.

In yet another embodiment, the cell culture media is supplemented withone or more amino acids in an amount effective to produce a low ARcomposition comprising about 15% or less AR2, 14% or less AR2, 13% orless AR2, 12% or less AR2, 11% or less AR2, 10% or less AR2, 9% or lessAR2, 8% or less AR2, 7% or less AR2, 6% or less AR2, 5% or less AR2,4.5% or less AR2, 4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5%or less AR2, 2% or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% orless AR2, 1.6% or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% orless AR2, 1.2% or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% orless AR2, 0.8% or less AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% orless AR2, 0.4% or less AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% orless AR2, or 0.0% AR2, and ranges within one or more of the preceding.

In another embodiment, the cell culture media is supplemented with oneor more amino acids in an amount effective to reduce the percentage ofacidic species in a protein or antibody composition by about 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, and ranges within one or more of the preceding.

In some embodiments, the one or more amino acids used to supplement thecell culture media is a basic amino acid. In certain embodiments the oneor more amino acids is arginine, lysine, histidine, ornithine, orcertain combinations of arginine or lysine with ornithine or of all fouramino acids. In certain embodiments, the amino acids are singlepeptides, as dipeptides, as tripeptides or as longer oligopeptides. Incertain embodiments, the di-, tri-, and/or oligopeptides areindividually composed of a single amino acid, while in alternativeembodiments, the di-, tri-, and/or oligopeptides are individuallycomposed of two or more particular amino acids. In certain embodiments,the amount of amino acid supplemented to the cell culture to achieveconcentrations of about 0 to about 9 g/L for arginine, about 0 to about11 g/L for lysine, about 0 to about 11 g/L histidine, and about 0 toabout 11 g/L ornithine. Wider ranges are also within the scope of theinstant invention, including, but not limited to: about 0 to about 30g/L for arginine, about 0 to about 30 g/L for lysine, about 0 to about30 g/L histidine, and about 0 to about 30 g/L ornithine.

For example, and not by way of limitation, as detailed in Example 1,below, when the production medium employed in the example wassupplemented with arginine to achieve a total concentration of 9 g/Larginine, the total amount of acidic species of adalimumab present in acell culture sample after purification was reduced from 19.7% of acontrol sample to 12.2% of the sample purified from the cells culturedwith the arginine supplemented media. Similarly, when the productionmedium employed in the example was supplemented with lysine, orhistidine, or ornithine to achieve total concentrations of 11 g/Llysine, 10 g/L ornithine or 10 g/L histidine, respectively, the totalamount of acidic species of adalimumab present in a cell culture sampleafter purification was reduced by 11.5%, 10.4% and 10.9%, respectively,compared to a control sample.

In certain embodiments, the cell culture media is supplemented, forexample, at the start of culture, or in a fed-batch or in a continuousmanner. The feed amounts may be calculated to achieve a certainconcentration based on offline or online measurements. The supplementsmay be added as multimers, e.g., arg-arg, his-his, arg-his-orn, etc.,and/or as chemical variants, e.g., of amino acids or analogs of aminoacids, salt forms of amino acids, controlled release of amino acids byimmobilizing in gels, etc, and/or in fully or partially dissolved form.The addition of one or more supplements may be based on measured amountof acidic species. The resulting media can be used in variouscultivation methods including, but not limited to, batch, fed-batch,chemostat and perfusion, and with various cell culture equipmentincluding, but not limited to, shake flasks with or without suitableagitation, spinner flasks, stirred bioreactors, airlift bioreactors,membrane bioreactors, reactors with cells retained on a solid support orimmobilized/entrapped as in microporous beads, and any otherconfiguration appropriate for optimal growth and productivity of thedesired cell line. In addition, the harvest criterion for these culturesmay be chosen, for example, based on choice of harvest viability orculture duration, to further optimize a certain targeted acidic speciesprofile.

Adjusting CaCl₂ and/or Niacinamide Concentration to Modulate AcidicSpecies (AR)

In certain embodiments, the cell culture media is supplemented withcalcium (e.g., as calcium chloride dihydrate) to achieve a calciumconcentration of between about 0.05 and 2.5 mM, or between about 0.05and 1 mM, or between about 0.1 and 0.8 mM, or between about 0.15 and 0.7mM, or between about 0.2 and 0.6 mM, or between about 0.25 and 0.5 mM,or between about 0.3 and 0.4 mM calcium.

In certain embodiments, the cell culture media is supplemented withcalcium (e.g., as calcium chloride dihydrate) in an amount effective toproduce a low AR composition comprising about 15% or less AR, 14% orless AR, 13% or less AR, 12% or less AR, 11% or less AR, 10% or less AR,9% or less AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or lessAR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR, 2.5%or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR, 1.7% orless AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR, 1.3% or lessAR, 1.2% or less AR, 1.1% or less AR, 1% or less AR, 0.9% or less AR,0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5% or less AR, 0.4%or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% or less AR, or 0.0%AR, and ranges within one or more of the preceding.

In another embodiment, the cell culture media is supplemented withcalcium (e.g., as calcium chloride dihydrate) in an amount effective toproduce a low AR composition comprising about 15% or less AR1, 14% orless AR1, 13% or less AR1, 12% or less AR1, 11% or less AR1, 10% or lessAR1, 9% or less AR1, 8% or less AR1, 7% or less AR1, 6% or less AR1, 5%or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% or less AR1, 3% orless AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or less AR1, 1.8% orless AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or less AR1, 1.4% orless AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or less AR1, 1% orless AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or less AR1, 0.6% orless AR1, 0.5% or less AR1, 0.4% or less AR1, 0.3% or less AR1, 0.2% orless AR1, 0.1% or less AR1, or 0.0% AR1, and ranges within one or moreof the preceding.

In yet another embodiment, the cell culture media is supplemented withcalcium (e.g., as calcium chloride dihydrate) in an amount effective toproduce a low AR composition comprising about 15% or less AR2, 14% orless AR2, 13% or less AR2, 12% or less AR2, 11% or less AR2, 10% or lessAR2, 9% or less AR2, 8% or less AR2, 7% or less AR2, 6% or less AR2, 5%or less AR2, 4.5% or less AR2, 4% or less AR2, 3.5% or less AR2, 3% orless AR2, 2.5% or less AR2, 2% or less AR2, 1.9% or less AR2, 1.8% orless AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5% or less AR2, 1.4% orless AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1% or less AR2, 1% orless AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% or less AR2, 0.6% orless AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% or less AR2, 0.2% orless AR2, 0.1% or less AR2, or 0.0% AR2, and ranges within one or moreof the preceding.

In another embodiment, the cell culture media is supplemented withcalcium (e.g., as calcium chloride dihydrate) in an amount effective toreduce the amount of acidic species in a protein or antibody sample byabout 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of thepreceding.

For example, and not by way of limitation, as detailed in Example 1,below, when the production medium employed in the example wassupplemented with calcium (e.g., as calcium chloride dihydrate) at aconcentration of 1.05 mM, the total amount of acidic species ofadalimumab present in a cell culture sample after purification wasreduced from 23.2% of a control sample to 16.5% of the sample purifiedfrom the cells cultured with the calcium supplemented media.

In certain embodiments, the cell culture can be supplemented with acombination of calcium, e.g., CaCl₂, and one or more a basic aminoacids, as described above. In certain embodiments, the amount of basicamino acid concentrations in combination with calcium in the cellculture is between about 0 to about 9 g/L for arginine, about 0 to about11 g/L for lysine, about 0 to about 11 g/L histidine, and about 0 toabout 11 g/L ornithine. Wider ranges are also within the scope of theinstant invention, including, but not limited to: about 0 to about 30g/L for arginine, about 0 to about 30 g/L for lysine, about 0 to about30 g/L histidine, and about 0 to about 30 g/L ornithine.

In certain embodiments, the cell culture media is supplemented withniacinamide to achieve a niacinamide concentration of between about 0.2and 3.0 mM, or between about 0.4 and 3.0 mM, or between about 0.8 and3.0 mM.

In some embodiments, the cell culture media is supplemented withniacinamide in an amount effective to reduce the amount of acidicspecies heterogeneity in a protein or antibody sample by about 15% orless AR, 14% or less AR, 13% or less AR, 12% or less AR, 11% or less AR,10% or less AR, 9% or less AR, 8% or less AR, 7% or less AR, 6% or lessAR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3%or less AR, 2.5% or less AR, 2% or less AR, 1.9% or less AR, 1.8% orless AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR, 1.4% or lessAR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1% or less AR,0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5%or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% orless AR, or 0.0% AR, and ranges within one or more of the preceding.

In another embodiment, the cell culture media is supplemented withniacinamide in an amount effective to produce a low AR compositioncomprising about 15% or less AR1, 14% or less AR1, 13% or less AR1, 12%or less AR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% orless AR1, 7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or lessAR1, 4% or less AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1,2% or less AR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1,1.6% or less AR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1,1.2% or less AR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1,0.8% or less AR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1,0.4% or less AR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1,or 0.0% AR1, and ranges within one or more of the preceding.

In yet another embodiment, the cell culture media is supplemented withniacinamide in an amount effective to produce a low AR compositioncomprising about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12%or less AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% orless AR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or lessAR2, 4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2,2% or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2,1.6% or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2,1.2% or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2,0.8% or less AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2,0.4% or less AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2,or 0.0% AR2, and ranges within one or more of the preceding.

For example, and not by way of limitation, as detailed in Example 1,below, when the production medium employed in the example wassupplemented with niacinamide at a concentration of 1.6 mM, the totalamount of acidic species of adalimumab present in a cell culture sampleafter purification was reduced from 19.9% of a control sample to 15.9%of the sample purified from the cells cultured with the niacinamidesupplemented media. In a separate example, where the media wassupplemented with 3 mM niacinamide, the total amount of acidic speciesof adalimumab present in a cell culture sample after purification wasreduced from 27.0% of a control sample to 19.8% of the sample purifiedfrom the cells cultured with the niacinamide supplemented media.

In certain embodiments, the cell culture can be supplemented with acombination of niacinamide, calcium, e.g., CaCl₂, and/or one or morebasic amino acids. In certain embodiments, the amount of basic aminoacid concentrations (after supplementation) in combination with calciumin the cell culture is between about 0 to about 9 g/L for arginine,about 0 to about 11 g/L for lysine, about 0 to about 11 g/L histidine,and about 0 to about 11 g/L ornithine. Although wider ranges are alsowithin the scope of the instant invention, including, but not limitedto: about 0 to about 30 g/L for arginine, about 0 to about 30 g/L forlysine, about 0 to about 30 g/L histidine, and about 0 to about 30 g/Lornithine.

In certain embodiments, the one or more amino acids, calcium, and/orniacinamide can be included in the medium at the start of culture, orcan be added in a fed-batch or in a continuous manner. The feed amountsmay be calculated to achieve a certain concentration based on offline oronline measurements. The addition of the supplement may be based onmeasured amount of acidic species. Other salts of particularsupplements, e.g., calcium, may also be used, for example calciumnitrate. The resulting media can be used in various cultivation methodsincluding, but not limited to, batch, fed-batch, chemostat andperfusion, and with various cell culture equipment including, but notlimited to, shake flasks with or without suitable agitation, spinnerflasks, stirred bioreactors, airlift bioreactors, membrane bioreactors,reactors with cells retained on a solid support or immobilized/entrappedas in microporous beads, and any other configuration appropriate foroptimal growth and productivity of the desired cell line.

In certain embodiments, a low AR composition is produced bysupplementing a clarified harvest. For example, but not by way oflimitation, such clarified harvests can be supplemented as describedabove (e.g., with calcium, niacinamide, and/or basic amino acids orcombinations thereof) to reduce AR formation (see Example 3).

Adjusting Process Parameters to Modulate Acidic Species (AR)

In certain embodiments, a low AR composition is produced by adjustmentof the dissolved oxygen (DO) concentration, and/or pH of the cellculture run. In certain embodiments, such adjustment includes increasingthe DO concentration of the cell culture, or decreasing the pH of thecell culture. Such increases in the DO concentration or decreases in thepH can be of a magnitude of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, andranges within one or more of the preceding, of the original amount.

In certain embodiments, cell cultures are run in DO concentrationsmaintained above about 15%, above about 20%, above about 30%, or betweenabout 15% and about 80%, between about 30% and about 50%, or at about80%, and ranges within one or more of the preceding, to achieve thedesired reduction in acidic species.

In certain embodiments, pH is either increased or decreased in order toincrease or decrease the amount of acidic species and/or the rate atwhich such acidic species form. For example, but not by way oflimitation, a reduction in pH to about 6.7 from a control pH of about7.1 can be employed to decrease the acidic species during cell cultureand the rate of acidic species formation in the context of a clarifiedharvest.

In certain embodiments, the DO concentration, and/or pH is maintained insuch a manner as to produce a low AR composition comprising about 15% orless AR, 14% or less AR, 13% or less AR, 12% or less AR, 11% or less AR,10% or less AR, 9% or less AR, 8% or less AR, 7% or less AR, 6% or lessAR, 5% or less AR, 4.5% or less AR, 4% or less AR, 3.5% or less AR, 3%or less AR, 2.5% or less AR, 2% or less AR, 1.9% or less AR, 1.8% orless AR, 1.7% or less AR, 1.6% or less AR, 1.5% or less AR, 1.4% or lessAR, 1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1% or less AR,0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% or less AR, 0.5%or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or less AR, 0.1% orless AR, or 0.0% AR, and ranges within one or more of the preceding.

In another embodiment, the DO concentration, and/or pH is maintained insuch a manner as to produce a low AR composition comprising about 15% orless AR1, 14% or less AR1, 13% or less AR1, 12% or less AR1, 11% or lessAR1, 10% or less AR1, 9% or less AR1, 8% or less AR1, 7% or less AR1, 6%or less AR1, 5% or less AR1, 4.5% or less AR1, 4% or less AR1, 3.5% orless AR1, 3% or less AR1, 2.5% or less AR1, 2% or less AR1, 1.9% or lessAR1, 1.8% or less AR1, 1.7% or less AR1, 1.6% or less AR1, 1.5% or lessAR1, 1.4% or less AR1, 1.3% or less AR1, 1.2% or less AR1, 1.1% or lessAR1, 1% or less AR1, 0.9% or less AR1, 0.8% or less AR1, 0.7% or lessAR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% or less AR1, 0.3% or lessAR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0% AR1, and ranges withinone or more of the preceding.

In yet another embodiment, the DO concentration, and/or pH is maintainedin such a manner as to produce a low AR composition comprising about 15%or less AR2, 14% or less AR2, 13% or less AR2, 12% or less AR2, 11% orless AR2, 10% or less AR2, 9% or less AR2, 8% or less AR2, 7% or lessAR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2, 4% or less AR2,3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2% or less AR2, 1.9%or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6% or less AR2, 1.5%or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2% or less AR2, 1.1%or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% or less AR2, 0.7% orless AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% or less AR2, 0.3% orless AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0% AR2, and rangeswithin one or more of the preceding.

In certain embodiments, the pH and/or DO is maintained in such a manneras to reduce the amount of acidic species in a protein or antibodysample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%,4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or moreof the preceding.

For example, and not by way of limitation, as detailed in Example 2,below, when five different pH conditions were assessed during cellculture: 7.1 7.0, 6.9, 6.8, and 6.7, the percent acidic speciesdecreased with a decrease in pH from 29.7% in the pH 7.1 condition to21.5% in the pH 6.7 condition, for a total reduction of 8.2%.

In addition, as detailed in Example 2, below (and not by way oflimitation), three different DO concentrations were assessed during cellculture: 20% DO concentration, 30% DO concentration and 50% DOconcentration, at 35° C. The percentage of acidic species was overalllower at higher DO concentrations. In particular, the percentage ofacidic species decreased with an increase in DO concentration from 23.9%in the 20% DO concentration sample to 20.3% in the 50% DO concentrationsample, for a total reduction of 3.6%.

In certain embodiments, a low AR composition is produced by cell culturecan be exerted by maintaining the DO concentration, and/or pH of thecell culture expressing the protein of interest as described hereinalong with choice of suitable temperature or temperature shiftstrategies, for example, but not limited to, lower process temperatureof operation, temperature shift to a lower temperature or a temperatureshift at an earlier culture time point. These culture conditions can beused in various cultivation methods including, but not limited to,batch, fed-batch, chemostat and perfusion, and with various cell cultureequipment including, but not limited to, shake flasks with or withoutsuitable agitation, spinner flasks, stirred bioreactors, airliftbioreactors, membrane bioreactors, reactors with cells retained on asolid support or immobilized/entrapped as in microporous beads, and anyother configuration appropriate for optimal growth and productivity ofthe desired cell line. These methods of modulating pH and/or DO and/ortemperature may also be used in combination with supplementation ofculture media with additives such as one or more amino acids,niacinamide, and/or calcium, or combinations thereof, as described aboveto maintain or achieve a target level of AR or to reduce the formationof AR during cell culture.

Continuous/Perfusion Cell Culture Technology to Modulate Acidic Species(AR)

In certain embodiments, a low AR composition is produced by the choiceof cell culture methodology. In certain embodiments, use of a continuousor perfusion technology may be utilized to achieve the desired loweringof acidic species in combination. In certain embodiments, this may beattained by modulation of medium exchange rate (where the exchange rateis the rate of exchange of medium in/out of a reactor).

In certain, non-limiting, embodiments, maintenance of the mediumexchange rates (working volumes/day) of a cell culture run between about0 and about 20, or between about 0.5 and about 12 or between about 1 andabout 8 or between about 1.5 and about 6 can be used to achieve thedesired reduction in acidic species.

For example, and not by way of limitation, as detailed in Example 4,below, when the medium exchange rate was chosen to be 1.5, the acidicspecies was 8.1%. With further increase in exchange rates to 6, afurther reduction in acidic species to 6% was obtained.

In certain embodiments, continuous or perfusion technology (e.g.,modulation of exchange rate) may result in a low AR compositioncomprising about 15% or less AR, 14% or less AR, 13% or less AR, 12% orless AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR,7% or less AR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or lessAR, 3.5% or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9%or less AR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% orless AR, 1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or lessAR, 1% or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR,0.6% or less AR, 0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2%or less AR, 0.1% or less AR, or 0.0% AR, and ranges within one or moreof the preceding.

In another embodiment, continuous or perfusion technology (e.g.,modulation of exchange rate) may result in a low AR compositioncomprising about 15% or less AR1, 14% or less AR1, 13% or less AR1, 12%or less AR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% orless AR1, 7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or lessAR1, 4% or less AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1,2% or less AR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1,1.6% or less AR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1,1.2% or less AR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1,0.8% or less AR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1,0.4% or less AR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1,or 0.0% AR1, and ranges within one or more of the preceding.

In yet another embodiment, continuous or perfusion technology (e.g.,modulation of exchange rate) may result in a low AR compositioncomprising about 15% or less AR2, 14% or less AR2, 13% or less AR2, 12%or less AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% orless AR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or lessAR2, 4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2,2% or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2,1.6% or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2,1.2% or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2,0.8% or less AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2,0.4% or less AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2,or 0.0% AR2, and ranges within one or more of the preceding.

In certain embodiments, continuous or perfusion technology (e.g.,modulation of exchange rate) may result in a low AR compositioncomprising about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%,4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or moreof the preceding.

In one embodiment, media containing additives, such as, for example, oneor more amino acids, calcium, and/or niacinamide, or combinationsthereof, as described above, may be used as the perfusion media tomaintain or achieve a target level of AR or to reduce the formation ofAR during cell culture.

In certain embodiments, a low AR composition is produced by, forexample, employment of an intermittent harvest strategy or through useof cell retention device technology.

IV. Preparation of Low AR Compositions Using Downstream ProcessTechnologies

In certain embodiments, the low AR compositions of the present inventionmay be produced using downstream process technologies (e.g.,purification), following cell culture of a protein. The downstreamprocess technologies may be used alone or in combination with theupstream process technologies described in Section III, above, and asdescribed in Example 10.

The methods described herein for the production of compositionscomprising low AR and/or low process-related impurities comprise thepurification of a protein, such as an antibody or antigen-bindingportion thereof, by, for example, chromatography, such as multimodal(MM) chromatography, wherein the MM media comprises both ion exchangeand hydrophobic interaction functional groups, and an aqueous saltsolution. In one embodiment, the same or substantially the same aqueoussalt solution is used as a loading buffer and a wash buffer.

In further embodiments, the methods described herein for the productionof compositions comprising low AR and/or low process-related impuritiescomprise the purification of a protein, such as an antibody orantigen-binding portion thereof, by chromatography comprising an anionexchange (AEX) resin and an aqueous salt solution. In one embodiment,the same or substantially the same aqueous salt solution is used as aloading buffer and a wash buffer.

In yet further embodiments, the methods described herein for theproduction of compositions comprising low AR and/or low process-relatedimpurities comprise the purification of a protein, such as an antibodyor antigen-binding portion thereof, by chromatography comprising acation exchange (CEX) adsorbent resin and an aqueous salt solution. Inone embodiment, the same or substantially the same aqueous salt solutionis used as a loading buffer and a wash buffer, and the target proteinbound to the CEX adsorbent resin is eluted with a buffer having a higherconductivity and/or pH than the loading/wash buffer.

In still further embodiments, the methods described herein forproduction of compositions comprising low AR and/or low process-relatedimpurities comprise the purification of a protein, such as an antibodyor antigen-binding portion thereof, by a combination of several media,for example by using an anion exchange (AEX) resin, and chromatographyusing a cation exchange (CEX) adsorbent resin, in a suitable buffer,such as, for example, a Tris/Formate buffer system. In one embodiment,the sample is purified affinity chromatography media, e.g., Protein A,prior to the ion chromatography resins. For example, in one embodiment,the methods described herein for production of compositions comprisinglow AR comprise the exemplary process reflected in FIG. 190.

In one embodiment, the method for producing a low AR compositioncomprising an antibody, or antigen binding portion thereof, comprisescontacting a first sample comprising the antibody, or antigen bindingportion thereof, to affinity chromatography media in a load buffer (forexample a low concentration Tris/Formate buffer), and eluting the samplefrom the affinity chromatography media as a first eluted sample,contacting the first eluted sample to a first chromatography media, suchas an AEX chromatography resin, in a load buffer, and eluting the samplefrom the AEX chromatography resin as a second eluted sample. The secondeluted sample is then contacted with a second chromatography media, suchas a CEX chromatography resin, in a load buffer, and the sample iseluted from the CEX chromatography resin as a third eluted sample. Inone embodiment, the CEX chromatography resin is eluted one, two, threeor more times. In one embodiment, the process optionally includes one ormore intermediate filtration steps, pH adjustment steps and/orinactivation steps.

In one embodiment, the downstream process technologies described herein,alone or in combination with other downstream process technologies orwith one or more upstream process technology, produce a low ARcomposition comprising an antibody, or antigen binding portion thereof,which contains 15% or less AR, 14% or less AR, 13% or less AR, 12% orless AR, 11% or less AR, 10% or less AR, 9% or less AR, 8% or less AR,7% or less AR, 6% or less AR, 5% or less AR, 4.5% or less AR, 4% or lessAR, 3.5% or less AR, 3% or less AR, 2.5% or less AR, 2% or less AR, 1.9%or less AR, 1.8% or less AR, 1.7% or less AR, 1.6% or less AR, 1.5% orless AR, 1.4% or less AR, 1.3% or less AR, 1.2% or less AR, 1.1% or lessAR, 1% or less AR, 0.9% or less AR, 0.8% or less AR, 0.7% or less AR,0.6% or less AR, 0.5% or less AR, 0.4% AR, 0.3% or less AR, 0.2% or lessAR, 0.1% or less AR, or 0.0% AR, and ranges within one or more of thepreceding. In one aspect of this embodiment, the low AR composition ofthe invention comprises about 0.0% to about 10% AR, about 0.0% to about5% AR, about 0.0% to about 4% AR, about 0.0% to about 3% AR, about 0.0%to about 2% AR, about 3% to about 5% AR, about 5% to about 8% AR, orabout 8% to about 10% AR, or about 10% to about 15% AR, and rangeswithin one or more of the preceding.

In one embodiment, the downstream process technologies described herein,alone or in combination with other downstream process technologies orwith one or more upstream process technology, produce a low ARcomposition comprising an antibody, or antigen binding portion thereof,which contains 15% or less AR1, 14% or less AR1, 13% or less AR1, 12% orless AR1, 11% or less AR1, 10% or less AR1, 9% or less AR1, 8% or lessAR1, 7% or less AR1, 6% or less AR1, 5% or less AR1, 4.5% or less AR1,4% or less AR1, 3.5% or less AR1, 3% or less AR1, 2.5% or less AR1, 2%or less AR1, 1.9% or less AR1, 1.8% or less AR1, 1.7% or less AR1, 1.6%or less AR1, 1.5% or less AR1, 1.4% or less AR1, 1.3% or less AR1, 1.2%or less AR1, 1.1% or less AR1, 1% or less AR1, 0.9% or less AR1, 0.8% orless AR1, 0.7% or less AR1, 0.6% or less AR1, 0.5% or less AR1, 0.4% orless AR1, 0.3% or less AR1, 0.2% or less AR1, 0.1% or less AR1, or 0.0%AR1, and ranges within one or more of the preceding. In one aspect ofthis embodiment, the low AR composition of the invention comprises about0.0% to about 10% AR1, about 0.0% to about 5% AR1, about 0.0% to about4% AR1, about 0.0% to about 3% AR1, about 0.0% to about 2% AR1, about 3%to about 5% AR1, about 5% to about 8% AR1, or about 8% to about 10% AR1,or about 10% to about 15% AR1, and ranges within one or more of thepreceding.

In one embodiment, the downstream process technologies described herein,alone or in combination with other downstream process technologies orwith one or more upstream process technology, produce a low ARcomposition comprising an antibody, or antigen binding portion thereof,which contains 15% or less AR2, 14% or less AR2, 13% or less AR2, 12% orless AR2, 11% or less AR2, 10% or less AR2, 9% or less AR2, 8% or lessAR2, 7% or less AR2, 6% or less AR2, 5% or less AR2, 4.5% or less AR2,4% or less AR2, 3.5% or less AR2, 3% or less AR2, 2.5% or less AR2, 2%or less AR2, 1.9% or less AR2, 1.8% or less AR2, 1.7% or less AR2, 1.6%or less AR2, 1.5% or less AR2, 1.4% or less AR2, 1.3% or less AR2, 1.2%or less AR2, 1.1% or less AR2, 1% or less AR2, 0.9% or less AR2, 0.8% orless AR2, 0.7% or less AR2, 0.6% or less AR2, 0.5% or less AR2, 0.4% orless AR2, 0.3% or less AR2, 0.2% or less AR2, 0.1% or less AR2, or 0.0%AR2, and ranges within one or more of the preceding. In one aspect ofthis embodiment, the low AR composition of the invention comprises about0.0% to about 10% AR2, about 0.0% to about 5% AR2, about 0.0% to about4% AR2, about 0.0% to about 3% AR2, about 0.0% to about 2% AR2, about 3%to about 5% AR2, about 5% to about 8% AR2, or about 8% to about 10% AR2,or about 10% to about 15% AR2, and ranges within one or more of thepreceding.

Protein Purification Generally

Following upstream processing of a protein of interest, downstreamprocess technologies can be used to purify the protein. For example, butnot by way of limitation, once a clarified solution or mixturecomprising the protein of interest, for example, an antibody or antigenbinding fragment thereof, has been obtained, separation of the proteinof interest from the acidic species can be effected using a combinationof different purification techniques, including, but not limited to,affinity separation steps, ion exchange separation steps, mixed modeseparation steps, and hydrophobic interaction separation stepssingularly or in combination. The separation steps separate mixtures ofproteins on the basis of their charge, degree of hydrophobicity, orsize, or any combination thereof, depending on the particular form ofseparation, including chromatographic separation. In one aspect of theinvention, separation is performed using chromatography, includingcationic, anionic, and hydrophobic interaction. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. Each of the separation methods result in the proteintraversing at different rates through a column, to achieve a physicalseparation that increases as they pass further through the column, oradhere selectively to the separation medium. The proteins are thendifferentially eluted by different elution buffers. In some cases, theantibody is separated from impurities when the impurities preferentiallyadhere to the column and the antibody less so, i.e., the desiredantibody variant is present in the Flow Through.

In certain embodiments, a low AR composition is produced usingchromatographic separation to identify the particular conditions, e.g.,salt concentration, pH, DO concentration, temperature, load amount andconditions, and washing conditions, sufficient to elicit the desiredfractionation profile, e.g., fractionation of acidic species and lysinevariants, of a sample comprising the protein of interest and at leastone process-related impurity. In certain embodiments, the method furthercomprises pooling the resulting fractions comprising the desired low ARcomposition compositions.

The purification process may begin at the separation step after theantibody has been produced using upstream production methods describedabove and/or by alternative production methods conventional in the art.Once a clarified solution or mixture comprising the protein of interest,e.g., an antibody, has been obtained, separation of the protein ofinterest from process-related impurities, such as the other proteinsproduced by the cell, as well as product-related substances, such acidicor basic variants, is performed. In certain non-limiting embodiments,such separation is performed using CEX, AEX, and/or MM chromatography.In certain embodiments, a combination of one or more differentpurification techniques, including affinity separation step(s), ionexchange separation step(s), mixed-mode step(s), and/or hydrophobicinteraction separation step(s) can also be employed. Such additionalpurification steps separate mixtures of proteins on the basis of theircharge, degree of hydrophobicity, and/or size. In one aspect of theinvention, such additional separation steps are performed usingchromatography, including hydrophobic, anionic or cationic interaction(or a combination thereof). Numerous chromatography resins arecommercially available for each of these techniques, allowing accuratetailoring of the purification scheme to the particular protein involved.Each of the separation methods allow proteins to either traverse atdifferent rates through a column, achieving a physical separation thatincreases as they pass further through the column, or to adhereselectively to a separation resin (or medium). The proteins are thendifferentially eluted using different eluents. In some cases, theprotein of interest is separated from impurities when the impuritiesspecifically adhere to the column's resin and the protein of interestdoes not, i.e., the protein of interest is contained in the effluent,while in other cases the protein of interest will adhere to the column'sresin, while the impurities and/or product-related substances areextruded from the column's resin during a wash cycle.

Primary Recovery and Virus Inactivation

In certain embodiments, the initial steps of the purification methods ofthe present invention involve the clarification and primary recovery ofantibody from a sample matrix. In certain embodiments, the primaryrecovery will include one or more centrifugation steps to separate theantibody product from the cells and cell debris. Centrifugation of thesample can be performed at, for example, but not by way of limitation,7,000×g to approximately 12,750×g. In the context of large scalepurification, such centrifugation can occur on-line with a flow rate setto achieve, for example, but not by way of limitation, a turbidity levelof 150 NTU in the resulting supernatant. Such supernatant can then becollected for further purification, or in-line filtered through one ormore depth filters for further clarification of the sample.

In certain embodiments, the primary recovery will include the use of oneor more depth filtration steps to clarify the sample matrix and therebyaid in purifying the antibodies of interest in the present invention. Inother embodiments, the primary recovery will include the use of one ormore depth filtration steps post centrifugation to further clarify thesample matrix. Non-limiting examples of depth filters that can be usedin the context of the instant invention include the Millistak+ X0HC,F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), Cuno™ model30/60ZA, 60/90 ZA, VR05, VR07, delipid depth filters (3M Corp.). A 0.2μm filter such as Sartorius's 0.45/0.2 μm Sartopore™ bi-layer orMillipore's Express SHR or SHC filter cartridges typically follows thedepth filters.

In certain embodiments, the primary recovery process can also be a pointat which to reduce or inactivate viruses that can be present in thesample matrix. For example, any one or more of a variety of methods ofviral reduction/inactivation can be used during the primary recoveryphase of purification including heat inactivation (pasteurization), pHinactivation, buffer/detergent treatment, UV and γ-ray irradiation andthe addition of certain chemical inactivating agents such asβ-propiolactone or e.g., copper phenanthroline as described in U.S. Pat.No. 4,534,972. In certain embodiments of the present invention, thesample matrix is exposed to detergent viral inactivation during theprimary recovery phase. In other embodiments, the sample matrix may beexposed to low pH inactivation during the primary recovery phase.

In those embodiments where viral reduction/inactivation is employed, thesample mixture can be adjusted, as needed, for further purificationsteps. For example, following low pH viral inactivation, the pH of thesample mixture is typically adjusted to a more neutral pH, e.g., fromabout 4.5 to about 8.5, prior to continuing the purification process.Additionally, the mixture may be diluted with water for injection (WFI)to obtain a desired conductivity.

Additives to the Clarified Harvest

In certain embodiments, a low AR composition is produced bysupplementing a clarified harvest containing antibodies or antigenbinding portions thereof. A clarified harvest can be extracted from acell culture, for example, a fermentation bioreactor, after undergoingcentrifugation to remove large solid particles and subsequent filtrationto remove finer solid particles and impurities from the material. Suchclarified harvests can be supplemented as described above (e.g., withcalcium, niacinamide, and/or basic amino acids, or combinations thereof)or modulation, e.g., lowering, of pH, to reduce AR formation (seeExample 3).

Affinity Chromatography

In certain embodiments, it will be advantageous to subject a sampleproduced by the techniques of the instant invention to affinitychromatography to further purify the protein of interest away fromacidic species. In certain embodiments the chromatographic material iscapable of selectively or specifically binding to the protein ofinterest (“capture”). Non-limiting examples of such chromatographicmaterial include: Protein A, Protein G, chromatographic materialcomprising, for example, an antigen bound by an antibody of interest,and chromatographic material comprising an Fc binding protein. Inspecific embodiments, the affinity chromatography step involvessubjecting the primary recovery sample to a column comprising a suitableProtein A resin. In certain embodiments, Protein A resin is useful foraffinity purification and isolation of a variety of antibody isotypes,particularly IgG1, IgG2, and IgG4. Protein A is a bacterial cell wallprotein that binds to mammalian IgGs primarily through their Fc regions.In its native state, Protein A has five IgG₁ binding domains as well asother domains of unknown function.

There are several commercial sources for Protein A resin. One suitableresin is MabSelect™ from GE Healthcare. Suitable resins include, but notlimited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelectXtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra,and ProSep Ultra Plus from EMD Millipore, MapCapture from LifeTechnologies. A non-limiting example of a suitable column packed withMabSelect™ is an about 1.0 cm diameter×about 21.6 cm long column (˜17 mLbed volume). This size column can be used for small scale purificationsand can be compared with other columns used for scale ups. For example,a 20 cm×21 cm column whose bed volume is about 6.6 L can be used forlarger purifications. Regardless of the column, the column can be packedusing a suitable resin such as MabSelect™

The Protein A column can be equilibrated with a suitable buffer prior tosample loading. Following the loading of the column, the column can bewashed one or multiple times using a suitable set of buffers. TheProtein A column can then be eluted using an appropriate elution buffer.For example, glycine-HCL or citric acid can be used as an elutionbuffer. The eluate can be monitored using techniques well known to thoseskilled in the art. The eluate fractions of interest can be collectedand then prepared for further processing.

The Protein A eluate may subject to a viral inactivation step either bydetergent or low pH, provided this step is not performed prior to theProtein A capture operation. A proper detergent concentration or pH andtime can be selected to obtain desired viral inactivation results. Afterviral inactivation, the Protein A eluate is usually pH and/orconductivity adjusted for subsequent purification steps.

The Protein A eluate may be subjected to filtration through a depthfilter to remove turbidity and/or various impurities from the antibodyof interest prior to additional chromatographic polishing steps.Examples of depth filters include, but not limited to, Millistak+X0HC,F0HC, D0HC, A1HC, and B1HC Pod filters (EMD Millipore), or Zeta Plus30ZA/60ZA, 60ZA/90ZA, delipid, VR07, and VR05 filters (3M). The ProteinA eluate pool may need to be conditioned to proper pH and conductivityto obtain desired impurity removal and product recovery from the depthfiltration step.

The invention is not limited to capture of the protein of interest usingProtein A chromatography. A non-Protein A chromatography capture stepcan also be carried out. For example, cation exchange capture andnon-chromatographic methods, such as aqueous two phase extraction orprecipitation, or other methods known in the art, can be used.

Anion Exchange Chromatography

In certain embodiments, the low AR compositions are produced bysubjecting the primary recovery sample to at least one anion exchangeseparation step. In certain embodiments, the anion exchange step willoccur after the above-described affinity chromatography, e.g., Protein Aaffinity, step.

The use of an anionic exchange material versus a cationic exchangematerial, such as those cation exchange materials discussed in detailbelow, is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy an anionic exchange step prior to the use of a cationic exchangestep, or a cationic exchange step prior to the use of an anionicexchange step. Furthermore, it is within the scope of this invention toemploy only an anionic exchange step, only an cationic exchange step, orany serial combination of the two (including serial combinations of oneor both ion exchange steps with the other chromatographic separationtechnologies described herein).

In performing the separation, the initial protein composition can becontacted with the anion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique.

For example, in the context of batch purification, anion exchangematerial is prepared in, or equilibrated to, the desired startingbuffer. Upon preparation, or equilibration, a slurry of the anionexchange material is obtained. The protein of interest, e.g., antibody,solution is contacted with the slurry to allow for protein adsorption tothe anion exchange material. The solution comprising the acidic speciesthat do not bind to the AEX material is separated from the slurry, e.g.,by allowing the slurry to settle and removing the supernatant. Theslurry can be subjected to one or more washing steps and/or elutionsteps.

In the context of chromatographic separation, a chromatographicapparatus, commonly cylindrical in shape, is employed to contain thechromatographic support material (e.g., AEX material) prepared in anappropriate buffer solution. The chromatographic apparatus, ifcylindrical, can have a diameter of about 5 mm to about 2 meters, and aheight of 5 cm to 50 cm, and in certain embodiments, particularly forlarge scale processing, a height of ≦30 cm is employed. Once thechromatographic material is added to the chromatographic apparatus, asample containing the protein of interest, e.g., an antibody, iscontacted to the chromatographic material to induce the separation. Anyportion of the solution that does not bind to the chromatographicmaterial, e.g., which may comprise, depending on the AEX material beingemployed, the protein of interest, acidic species, is separated from thechromatographic material by washing the material and collectingfractions from column. The chromatographic material can be subjected toone or more wash steps. If desired, the chromatographic material canthen be contacted with a solution designed to desorb any components ofthe solution that have bound to the chromatographic material.

In certain embodiments, a wash step can be performed in the context ofAEX chromatography using conditions similar to the load conditions oralternatively by decreasing the pH and/or increasing the ionicstrength/conductivity of the wash in a step wise or linear gradientmanner. The resulting Flow Through and wash fractions can be analyzedand appropriate fractions pooled to achieve the desired reduction incharged variant species. In certain embodiments, the aqueous saltsolution used as both the loading and wash buffer has a pH that at ornear the isoelectric point (pI) of the protein of interest. In certainembodiments the pH is about 0 to 2 units higher or lower than the pI ofthe protein of interest. In certain embodiments, it will be in the rangeof 0 to 0.5 units higher or lower. In certain embodiments, it will be atthe pI of the antibody.

In certain non-limiting embodiments, the anionic agent is selected fromthe group consisting of acetate, formate, or combinations thereof. Incertain non-limiting embodiments, the cationic agent is selected fromthe group consisting of Tris, arginine, or combinations thereof. In oneembodiment, the buffer solution is a Tris/formate buffer. In anotherembodiment, the buffer is selected from the group consisting ofpyridine, piperazine, L-histidine, Bis-tris, Bis-tris propane,imidazole, N-Ethylmorpholine, TEA (triethanolamine), Tris, Morpholine,N-Methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol),diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol),piperazine, 1,3-Diaminopropane, piperidine

A packed anion-exchange chromatography column, anion-exchange membranedevice, anion-exchange monolithic device, or depth filter media can beoperated either in bind-elute mode, flow-through mode, or a hybrid modewherein the product exhibits binding to the chromatographic material,yet can be washed from the column using a buffer that is the same orsubstantially similar to the loading buffer. In the bind-elute mode, thecolumn or the membrane device is first conditioned with a buffer withappropriate ionic strength and pH under conditions where certainproteins will be immobilized on the resin based matrix. For example, incertain embodiments, during the feed load, the protein of interest willbe adsorbed to the resin due to electrostatic attraction. After washingthe column or the membrane device with the equilibration buffer oranother buffer with different pH and/or conductivity, the productrecovery is achieved by increasing the ionic strength (i.e.,conductivity) of the elution buffer to compete with the solute for thecharged sites of the anion exchange matrix. Changing the pH and therebyaltering the charge of the solute is another way to achieve elution ofthe solute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution). In the flow-through mode, thecolumn or the membrane device is operated at selected pH andconductivity such that the protein of interest does not bind to theresin or the membrane while the acidic species will either be retainedon the column or will have a distinct elution profile as compared to theprotein of interest. In the context of this hybrid strategy, acidicspecies will bind to the chromatographic material (or Flow Through) in amanner distinct from the protein of interest, e.g., while the protein ofinterest and certain aggregates and/or fragments of the protein ofinterest may bind the chromatographic material, washes thatpreferentially remove the protein of interest can be applied. The columnis then regenerated before next use.

Non-limiting examples of anionic exchange substituents includediethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternaryamine (Q) groups. Additional non-limiting examples include: Poros 50PIand Poros 50HQ, which are a rigid polymeric bead with a backboneconsisting of cross-linked poly[styrene-divinylbenzene]; Capto Q Impresand Capto DEAE, which are a high flow agarose bead; Toyopearl QAE-550,Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are a polymericbase bead; Fractogel® EMD TMAE Hicap, which is a synthetic polymericresin with a tentacle ion exchanger; Sartobind STIC® PA nano, which is asalt-tolerant chromatographic membrane with a primary amine ligand;Sartobind Q nano; which is a strong anion exchange chromatographicmembrane; CUNO BioCap; which is a zeta-plus depth filter mediaconstructed from inorganic filter aids, refined cellulose, and an ionexchange resin; and X0HC, which is a depth-filter media constructed frominorganic filter aid, cellulose, and mixed cellulose esters. Thedetailed information is listed in Table 1.

TABLE 1 List of AEX Adsorbent Properties Media Particle/ AEX AdsorbentVendor Type Ligand Type Pore Size Catalog Number Poros PI Applied ResinWeak ~50 μm 1-2459-11 Poros HQ Biosystems Strong ~50 μm 1-2559-11 CaptoDEAE GE Weak ~90 μm 17-5443-10 CaptoQ Impres Strong ~90 μm 17-5316-10QAE-550 Tosoh Strong ~100 μm 43271 DEAE-650 Weak ~65 μm 43201 GigaCapQ-650 Strong ~75 μm 21854 TMAE HiCap EMD/Millipore Strong ~40-90 μm1.16881.0013 Sartobind Sartorius Membrane Weak 3-5 μm 92STPA42DN- STIC ®PA Nano 11-A Sartobind Q Strong 3-5 μm 92IEXQ42DN-11 Nano CUNO BioCap 3MDepth NA NA BC0025L60ZA05A 25 Filter X0HC Millipore NA NA MX0HC23CL3

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 50 and 500 g/L, or between about 75 and 350 g/L, orbetween about 200 and 300 g/L. In certain embodiments, the proteinconcentration of the load protein mixture is adjusted to a proteinconcentration of the material loaded to the column of about 0.5 and 50g/L, between about 1 and 20 g/L, or between 3 and 10 g/L. In certainembodiments, the protein concentration of the load protein mixture isadjusted to a protein centration of the material to the column of about37 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR in the Flow Through and wash fractions while enriching for the samein the flow elution fraction, thereby producing protein compositionsthat have reduced AR or are free of AR. In certain embodiments relatingto but not limited to adalimumab, the methods of the instant inventioncan be used to selectively remove, significantly reduce, or essentiallyremove all of AR1 charge variants in the Flow Through and wash fractionswhile enriching for the same in the flow elution fraction, therebyproducing protein compositions that have reduced AR1 or are free of AR1variants. In certain embodiments relating to but not limited toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR2 charge variants in the flow-through and wash fractions whileenriching for the same in the flow elution fraction, thereby producingprotein compositions that have reduced AR2 or are free of AR2 variants.

In certain embodiments, including but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe MGO variants in the Flow Through and wash fractions while enrichingfor the same in the elution fraction, thereby producing proteincompositions that have reduced MGO or are free of MGO variants (forexample, see U.S. Patent Application Ser. No. 61/777,883, filed on Mar.12, 2013). In certain embodiments, including, but not limited to thoserelating to adalimumab, the methods of the instant invention can be usedto selectively remove, significantly reduce, or essentially remove allof the glycated variants (Schiff's base and permanently glycated forms)in the Flow Through and wash fractions while enriching for the same inthe elution fraction, thereby producing protein preparations withreduced or free of glycated variants.

In certain embodiments, the loading, pH, conductivity of the AEXchromatography step, as well as elution pH conductivity, can be modifiedto achieve a desired distribution of product-relates substances (AR orlysine variants) For example, but not by way of limitation, certainembodiments are directed to the modulation of the lysine distribution ofpurified sample of a protein of interest, e.g., increasing Lys 0 anddecreasing Lys 1 and Lys 2. In certain embodiments, the methods of thepresent invention allow for the preparation of samples wherein theamount of Lys 0 is decreased, while the amount of Lys 1 and/or Lys 2 isincreased.

In certain embodiments, an AEX chromatographic separation can beperformed and combinations of fractions can be pooled to achieve acombination of desired process-related impurity and/or product-relatessubstance levels, in addition to, or in place of merely modulatingcharge variant concentration.

Spectroscopy methods such as UV, NIR, FTIR, Fluorescence, and Raman maybe used to monitor levels of AR species in an on-line, at-line orin-line mode, which can then be used to control the level of chargevariants, e.g., acidic species, in the pooled material collected fromthe AEX effluent.

In certain embodiments, specific signals arising from the chemicalmodification of the proteins such as glycation, MGO modification,deamidation, glycosylation may be specifically measurable byspectroscopic methods through such in-line, on-line or at-line methods,enabling realtime or near-real time control of product quality of theresulting product. In certain embodiments, on-line, at-line or in-linemonitoring methods can be used either on the effluent line of thechromatography step or in the collection vessel, to enable achievementof the desired product quality/recovery. In certain embodiments, the UVsignal can be used as a surrogate to achieve an appropriate productquality/recovery, wherein the UV signal can be processed appropriately,including, but not limited to, such processing techniques asintegration, differentiation, moving average, such that normal processvariability can be addressed and the target product quality can beachieved. In certain embodiments, such measurements can be combined within-line dilution methods such that ion concentration/conductivity of theload/wash can be controlled by feedback and hence facilitate productquality control.

In certain embodiments, a combination of AEX, CEX and/or MM methods canbe used to prepare product-related substance-modulated materials,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, and/or UF/DFsteps so as to achieve the desired AR, product quality, ionconcentration, and/or viral reduction.

As described below and in Example 11, AEX chromatography can be used inconjunction with recycle chromatography modes and continuouschromatography modes.

Cation Exchange Chromatography

The low AR compositions of the present invention can be produced bysubjecting the composition, e.g., a primary recovery sample, to at leastone cation exchange separation step. In certain embodiments, the CEXstep will occur after the above-described affinity chromatography, e.g.,Protein A affinity, step.

The use of a cationic exchange material versus an anionic exchangematerial, such as those anionic exchange materials discussed in detailabove, is based on the local charges of the protein of interest in agiven solution. Therefore, it is within the scope of this invention toemploy a cationic exchange step prior to the use of an anionic exchangestep, or an anionic exchange step prior to the use of a cationicexchange step. Furthermore, it is within the scope of this invention toemploy only a cationic exchange step, only an anionic exchange step, orany serial combination of the two (including serial combinations of oneor both ion exchange steps with the other chromatographic separationtechnologies described herein).

In performing the separation, the initial protein mixture can becontacted with the cation exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique, as described above in connection with ProteinA or AEX.

In certain embodiments, the aqueous salt solution used as both theloading and wash buffer has a pH that is lower than the isoelectricpoint (pI) of the protein of interest. In certain embodiments, the pH isabout 0 to 5 units lower than the pI of the protein. In certainembodiments, it is in the range of 1 to 2 units lower. In certainembodiments, it is in the range of 1 to 1.5 units lower.

In certain embodiments, the concentration of the anionic agent inaqueous salt solution is increased or decreased to achieve a pH ofbetween about 3.5 and 10.5, or between about 4 and 10, or between about4.5 and 9.5, or between about 5 and 9, or between about 5.5 and 8.5, orbetween about 6 and 8, or between about 6.5 and 7.5. In certainembodiments, the concentration of anionic agent is increased ordecreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or6, or 6.5, or 6.8, or 7.5. Buffer systems suitable for use in the CEXmethods include, but are not limited to tris formate, tris acetate,ammonium sulfate, sodium chloride, and sodium sulfate.

In certain embodiments, the conductivity and pH of the aqueous saltsolution is adjusted by increasing or decreasing the concentration of acationic agent. In certain embodiments, the cationic agent is maintainedat a concentration of between about range of 20 mM to 500 mM, or betweenabout 50 to 350 mM or between about 100 to 300 mM or between about 100to 200 mM.

In certain non-limiting embodiments, the cationic agent is selected fromthe group consisting of sodium, Tris, tromethalmine, ammonium, arginine,or combinations thereof. In certain non-limiting embodiments, theanionic agent is selected from the group consisting of formate, acetate,citrate, chloride anion, sulphate, phosphate or combinations thereof.

A packed cation-exchange chromatography column or a cation-exchangemembrane device can be operated either in bind-elute mode, flow-throughmode, or a hybrid mode wherein the product exhibits binding to thechromatographic material, yet can be washed from the column using abuffer that is the same or substantially similar to the loading buffer.The details of these modes are outlined above.

Cationic substituents include carboxymethyl (CM), sulfoethyl (SE),sulfopropyl (SP), phosphate (P) and sulfonate (S). Additional cationicmaterials include, but are not limited to: Capto SP ImpRes, which is ahigh flow agarose bead; CM Hyper D grade F; which is a ceramic beadcoated and permeated with a functionalized hydrogel, 250-400 ionicgroups μeq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether basematrix with 50-100 μeq/mL ionic capacity; Nuvia C Prime, which is ahydrophobic cation exchange media composed of a macroporous highlycrosslinked hydrophilic polymer matrix 55-75 μeq/mL; Nuvia S, which hasa UNOsphere base matrix with 90-150 μeq/mL ionic groups; Poros HS; whichis a rigid polymetic bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene]; Poros XS; which is a rigid polymetic beadwith a backbone consisting of cross-linked poly[styrene-divinylbenzene];Toyo Pearl Giga Cap CM 650M, which is a polymeric base bead with 0.225meq/mL ionic capacity; Toyo Pearl Giga Cap S 650M which is a polymericbase bead; Toyo Pearl MX TRP, which is a polymeric base bead. Detailedinformation concerning the aforementioned materials is listed in Table2. It is noted that CEX chromatography can be used with MM resins,described herein.

TABLE 2 Cationic Materials Catalog Resin Vendor type particle sizeNumber Capto SP ImpRes GE Strong ~40 μm 17-5468-10 CM Hyper D Pall Weak~50 μm 20050-027 Eshmuno S Millipore Strong ~85 μm 1.20078 Nuvia C PrimeBiorad Mix ~70 μm 156-3401 Mode Nuvia S Biorad Strong ~85 μm 156-0315Poros HS Applied Weak ~50 μm 13359-06 Biosystems Poros XS Applied Strong~50 μm 4404337 Biosystems Toyo Pearl Giga Tosoh Weak ~75 μm 21946 Cap CM650M Toyo Pearl Giga Tosoh Strong ~75 μm 21833 Cap S 650M Toyo PearlTosoh Mix ~75 μm 22817 MX Trp 650M Mode

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 5 and 150 g/L, or between about 10 and 100 g/L, betweenabout 20 and 80 g/L, between about 30 and 50 g/L, or between about 40and 50 g/L. In certain embodiments, the protein concentration of theload protein mixture is adjusted to a protein concentration of thematerial loaded to the column of about 0.5 and 50 g/L, or between about1 and 20 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR in the Flow Through and wash fractions while enriching for the samein the elution fraction, thereby producing protein compositions thathave reduced AR or are free of AR. In certain embodiments relating tobut not limited to adalimumab, the methods of the instant invention canbe used to selectively remove, significantly reduce, or essentiallyremove all of AR1 charge variants in the Flow Through and wash fractionswhile enriching for the same in the flow elution fraction, therebyproducing protein compositions that have reduced AR1 or are free of AR1variants. In certain embodiments relating to but not limited toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR2 charge variants in the flow-through and wash fractions whileenriching for the same in the flow elution fraction, thereby producingprotein compositions that have reduced AR2 or are free of AR2 variants.

In certain embodiments, including, but not limited to those relating toadalimumab, the methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe MGO variants in the elution fractions while enriching for the samein the Flow Through and wash fractions, thereby producing proteinpreparations with reduced or free of MGO variants. In certainembodiments, including, but not limited to those relating to adalimumab,the methods of the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of the glycated variants(Schiff's base and permanently glycated forms) in the elution fractionswhile enriching for the same in the Flow Through and wash fractions,thereby producing protein preparations with reduced or free of glycatedvariants.

In certain embodiments, the loading, pH, conductivity of the CEXchromatography step, as well as elution pH conductivity, can be modifiedto achieve a desired distribution of acidic species. For example, butnot by way of limitation, certain embodiments are directed to themodulation of the lysine distribution of a purified sample of a proteinof interest, e.g., increasing Lys 0 and decreasing Lys 1 and Lys 2. Incertain embodiments, the methods of the present invention allow for thepreparation of samples wherein the amount of Lys 0 is decreased, whilethe amount of Lys 1 and/or Lys 2 is increased.

In certain embodiments, a CEX chromatographic separation can beperformed and combinations of fractions can be pooled to achieve acombination of desired process-related impurity and/or product-relatessubstance levels, in addition to, or in place of merely modulatingcharge variant concentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of product-relatedcharge variants, aggregates, low molecular weight variants (e.g.,fragments of the protein of interest) in an on-line, at-line or in-linemode, which can then be used to control the level of charge variants,e.g., acidic species, in the pooled material collected from the CEXeffluent. In certain embodiments, specific signals arising from thechemical modification of the proteins such as glycation, MGOmodification, deamidation, glycosylation may be specifically measurableby spectroscopic methods through such in-line, on-line or at-linemethods, enabling realtime or near-real time control of product qualityof the resulting product. In certain embodiments, on-line, at-line orin-line monitoring methods can be used either on the effluent line ofthe chromatography step or in the collection vessel, to enableachievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

In certain embodiments, a combination of CEX and AEX and/or MM methodscan be used to prepare product-related substance-modulated materials,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalchromatography, filtration, pH adjustment, UF/DF steps so as to achievethe desired product quality, AR, ion concentration, and/or viralreduction.

As described below and in Example 11, CEX chromatography can be used inconjunction with recycle chromatography and continuous chromatographymodes.

Mixed Mode Chromatography

Mixed mode (“MM”) chromatography may also be used to prepare the low ARcompositions of the invention. MM chromatography, also referred toherein as “multimodal chromatography”, is a chromatographic strategythat utilizes a support comprising a ligand that is capable of providingat least two different, and in certain embodiments co-operative, sitesthat interact with the substance to be bound. In certain embodiments,one of these sites gives an attractive type of charge-charge interactionbetween the ligand and the substance of interest and the other siteprovides for electron acceptor-donor interaction and/or hydrophobicand/or hydrophilic interactions. Electron donor-acceptor interactionsinclude interactions such as hydrogen-bonding, π-π, cation-π, chargetransfer, dipole-dipole, induced dipole etc.

In certain embodiments, the resin employed for a mixed mode separationis Capto Adhere. Capto Adhere is a strong anion exchanger withmultimodal functionality. Its base matrix is a highly cross-linkedagarose with a ligand (N-Benzyl-N-methyl ethanol amine) that exhibitsmany functionalities for interaction, such as ionic interaction,hydrogen bonding and hydrophobic interaction. In certain embodiments,the resin employed for a mixed mode separation is selected fromPPA-HyperCel and HEA-HyperCel. The base matrices of PPA-HyperCel andHEA-HyperCel are high porosity cross-linked cellulose. Their ligands arePhenylpropylamine and Hexylamine, respectively. Phenylpropylamine andHexylamine offer different selectivity and hydrophobicity options forprotein separations. Additional mixed mode chromatographic supportsinclude, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M,and Eshmuno® HCX.

In certain embodiments, the mixed mode chromatography resin is comprisedof ligands coupled to an organic or inorganic support, sometimes denoteda base matrix, directly or via a spacer. The support may be in the formof particles, such as essentially spherical particles, a monolith,filter, membrane, surface, capillaries, etc. In certain embodiments, thesupport is prepared from a native polymer, such as cross-linkedcarbohydrate material, such as agarose, agar, cellulose, dextran,chitosan, konjac, carrageenan, gellan, alginate etc. To obtain highadsorption capacities, the support can be porous, and ligands are thencoupled to the external surfaces as well as to the pore surfaces. Suchnative polymer supports can be prepared according to standard methods,such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta79(2), 393-398 (1964). Alternatively, the support can be prepared from asynthetic polymer, such as cross-linked synthetic polymers, e.g. styreneor styrene derivatives, divinylbenzene, acrylamides, acrylate esters,methacrylate esters, vinyl esters, vinyl amides etc. Such syntheticpolymers can be produced according to standard methods, see e.g.“Styrene based polymer supports developed by suspension polymerization”(R Arshady: Chimica e L′Industria 70(9), 70-75 (1988)). Porous native orsynthetic polymer supports are also available from commercial sources,such as Amersham Biosciences, Uppsala, Sweden.

In certain embodiments, the protein load of the mixture comprisingprotein of interest is adjusted to a total protein load to the column ofbetween about 50 and 750 g/L, or between about 75 and 500 g/L, orbetween about 100 and 300 g/L. In certain embodiments, the proteinconcentration of the load protein mixture is adjusted to a proteinconcentration of the material loaded to the column of about 1 and 50g/L, or between about 9 and 25 g/L.

In certain embodiments, additives such as poly ethylene glycol,detergents, amino acids, sugars, chaotropic agents can be added toenhance the performance of the separation, so as to achieve betterrecovery or product quality.

In certain embodiments, including, but not limited to those relating toadalimumab, the MM methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofAR in the Flow Through and wash fractions while enriching for the samein the flow elution fraction, thereby producing protein compositionsthat have reduced AR or are free of AR. In certain embodimentsincluding, but not limited to those relating to adalimumab, the methodsof the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of AR1 charge variantsin the Flow Through and wash fractions while enriching for the same inthe flow elution fraction, thereby producing protein compositions thathave reduced AR1 or are free of AR1 variants. In certain embodimentsincluding, but not limited to those relating to adalimumab, the methodsof the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of AR2 charge variantsin the flow-through and wash fractions while enriching for the same inthe flow elution fraction, thereby producing protein compositions thathave reduced AR2 or are free of AR2 variants.

In certain embodiments, including, but not limited to those relating toadalimumab, the MM methods of the instant invention can be used toselectively remove, significantly reduce, or essentially remove all ofthe MGO variants in the Flow Through and wash fractions while enrichingfor the same in the elution fraction, thereby producing proteinpreparations with reduced or free of MGO variants. In certainembodiments, including, but not limited to those relating to adalimumab,the methods of the instant invention can be used to selectively remove,significantly reduce, or essentially remove all of the glycated variants(Schiff's base and permanently glycated forms) in the Flow Through andwash fractions while enriching for the same in the elution fraction,thereby producing protein preparations with reduced or free of glycatedvariants.

In certain embodiments, the loading, pH, conductivity of the MMchromatography step, wash pH and conductivity, as well as elution pHconductivity, can be modified to achieve a desired distribution ofacidic species. For example, but not by way of limitation, certainembodiments are directed to the modulation of the lysine distribution ofa purified sample of a protein of interest, e.g., increasing Lys 0 anddecreasing Lys 1 and Lys 2. In certain embodiments, the methods of thepresent invention allow for the preparation of samples wherein theamount of Lys0 is decreased, while the amount of Lys 1 and/or Lys 2 isincreased.

In certain embodiments, a MM chromatographic separation can be performedand combinations of fractions can be pooled to achieve a combination ofdesired process-related impurity and/or product-relates substancelevels, in addition to, or in place of merely modulating charge variantconcentration.

In certain embodiments, spectroscopy methods such as UV, NIR, FTIR,Fluorescence, Raman may be used to monitor levels of AR species in anon-line, at-line or in-line mode, which can then be used to control thelevel of charge variants, e.g., acidic species, in the pooled materialcollected from the MM effluent. In certain embodiments, specific signalsarising from the chemical modification of the proteins such asglycation, MGO modification, deamidation, glycosylation may bespecifically measurable by spectroscopic methods through such in-line,on-line or at-line methods, enabling realtime or near-real time controlof product quality of the resulting product. In certain embodiments,on-line, at-line or in-line monitoring methods can be used either on theeffluent line of the chromatography step or in the collection vessel, toenable achievement of the desired product quality/recovery. In certainembodiments, the UV signal can be used as a surrogate to achieve anappropriate product quality/recovery, wherein the UV signal can beprocessed appropriately, including, but not limited to, such processingtechniques as integration, differentiation, moving average, such thatnormal process variability can be addressed and the target productquality can be achieved. In certain embodiments, such measurements canbe combined with in-line dilution methods such that ionconcentration/conductivity of the load/wash can be controlled byfeedback and hence facilitate product quality control.

In certain embodiments, a combination of MM and AEX and/or CEX methodscan be used to prepare the low AR compositions of the invention,including certain embodiments where one technology is used in acomplementary/supplementary manner with another technology. In certainembodiments, such a combination can be performed such that certainsub-species are removed predominantly by one technology, such that thecombination provides the desired final composition/product quality. Incertain embodiments, such combinations include the use of additionalintervening chromatography, filtration, pH adjustment, UF/DF steps so asto achieve the desired product quality, AR, ion concentration, and/orviral reduction.

As described below and in Example 11, MM chromatography can be used inconjunction with recycle chromatography and continuous chromatographymodes.

Continuous and Recycle Chromatography

Continuous and recycle chromatography modes can be used to produce thelow AR compositions of the invention, and are described below and IinExample 11. These methods result in significant improvements in recoveryof the protein, e.g., antibody, of interest while maintaining the ARreduction levels. These continuous and recycle chromatography modes areapplicable to chromatography methods where (a) the low acidic speciescomponent of interest is collected in the unbound fraction during thechromatography (Flow Through/wash chromatography) or (b) where the lowacidic species component of interest is first bound to the media andsubsequently recovered by washing the media with conditions that elutethe bound component.

Continuous and Recycle Chromatography—Flow Through/Wash Chromatography

In the case where the low acidic species component of interest iscollected in the unbound fraction, the following approach may beemployed which prevents loss of the material loaded on the column.

In one embodiment, a recycle chromatography mode may be used wherein thecolumn is loaded and the unbound fractions that result in the target ARlevel are collected. Subsequently, instead of regenerating the columnand losing the product, the column may be washed under conditions thatresult in recovery of the product remaining bound to the column. In oneembodiment, the product recovered under these conditions containssignificantly higher AR levels than the original feed material. In oneembodiment, this wash fraction may be adjusted to the appropriateconditions to achieve the separation desired on subsequent processing(typically similar conditions to the initial preparation) and combinedwith the original feed material and loaded on the column again (afterpreparing the column appropriately for the next cycle). In oneembodiment, the amount of material prepared for the next cycle,combining the wash fraction from the first cycle and the fresh material,may be adjusted to the target loading capacity for the column to achievethe desired separation (typically similar to the capacity targeted forthe first cycle).

In one embodiment, in performing the second cycle, a similar strategymay be employed, collecting the unbound fraction so as to achieve thetarget AR level and then subsequently washing the column underconditions to recover the product remaining on the column.

In one embodiment, this recycle chromatography mode is continued untilall the load materials are used. The number of cycles can be controlledby designing the column size appropriately.

In employing the recycle chromatography mode, the recovery of theproduct loaded on the column may be significantly improved whileachieving the target AR levels.

Several variations of the recycle chromatography mode can be employed.In one embodiment, the fractions that are collected targeting a certainAR level can be determined based on predetermined criteria or based onat-line, off-line or on-line analysis of the effluent of the column orthe collected pool.

In another embodiment, the wash conditions used for the first cycle canbe adjusted to recover the desired amount of product at the desiredproduct quality, only limited by the feasibility of preparing anappropriate load mixture for the subsequent step. In one aspect of thisembodiment, the wash condition may be similar to the load condition. Inanother aspect of this embodiment, the wash condition can be stringentto recover all of the product species (desired and undesired) remainingon the column.

In still another embodiment, the loading amount, the loading conditionsand the washing conditions used for the subsequent cycles can bemodified to achieve the desired purity, given that that loading materialfor the subsequent cycles are likely to contain higher levels of AR.

In another embodiment, the last cycle of the operation can be performedunder different conditions such that the target purity and targetrecovery can be achieved to optimize overall recovery and purity.

The methods for producing the low AR composition of the invention canalso be implemented in a continuous chromatography mode. In this mode,at least two columns may be employed (referred to as a “first” columnand a “second” column). In one embodiment, the feed material may beloaded onto the first column, and the unbound fraction from the firstcolumn may be collected such that the pool material contains the targetAR level. In one embodiment, the column may be then washed underconditions that recover the remaining product. In one embodiment, thismaterial may be then dynamically diluted with appropriate solutions toachieve the desired loading conditions, mixed with fresh feed materialand directed to the second column. In one embodiment, the unboundfraction from the second column may be collected to achieve the targetAR level. The second column may be then washed under conditions torecover the product and diluted with appropriate solutions, mixed withfresh materials dynamically and directed to the first column (which isprepared to receive the load after regeneration/cleaning). In oneembodiment, this cycling is continued until all the load material isused. The last cycle can be operated in a “typical” mode, withappropriate adjustments to the load and wash conditions as necessary.

In certain embodiments, this continuous chromatography mode can becarried out such that the wash material containing the higher AR levelscan be directed back into the load tank after appropriate dilution. Thismaterial can then be loaded subsequently or concurrently onto the secondcolumn, such that the operation of the two columns are not in tandem,reducing complexity of the operation.

This continuous chromatography mode, while similar to the recyclechromatography mode, can be carried out more efficiently, and thereforehas a reduced processing time in some embodiments.

For this continuous chromatography mode, several variations can beemployed. In one embodiment, the fractions that are collected targetinga certain AR level can be determined based on predetermined criteria orbased on at-line, off-line or on-line analysis of the effluent of thecolumn or the collected pool.

In another embodiment, the wash conditions used for the first cycle canbe adjusted to recover the desired amount of product at the desiredproduct quality, only limited by the feasibility of preparing anappropriate load mixture for the subsequent step. In one aspect of thisembodiment, the wash conditions may be similar to the load conditions.In another aspect of the embodiment, the wash conditions can bestringent to recover all of the product species (desired and undesired)remaining on the column.

In still another embodiment, the loading amount, the loading conditionsand the washing conditions used for the subsequent cycles can bemodified to achieve the desired purity, given that that loading materialfor the subsequent cycles are likely to contain higher levels of AR.

In another embodiment, the last cycle of the operation can be performedunder different conditions such that the target purity/recovery can beachieved to optimize overall recovery and and/or purity.

In one embodiment, the media choice for the recycle or continuous modescan be one of many chromatographic resins with pendant hydrophobic andanion exchange functional groups, monolithic media, membrane adsorbentmedia or depth filtration media.

In certain embodiments, membrane or depth filter based media(“convective media”) can be used in the recycle or continuouschromatography modes because selectivity of separation is not requiredto be high given the fact that the less enriched portions of the productare “recycled” while the pure fractions are selectively pooled.

Continuous and Recycle Chromatography—Elution Chromatography

In the elution mode of chromatography or separation, as exemplified bythe CEX technology for AR reduction, the conditions are chosen for theload and wash steps such that the AR enriched material is collected inthe Flow Through and/or wash fractions, while the AR reduced material iscollected in the elution fraction. In the typical implementation of theCEX technology, about 10 to 40% of the product (the desired chargevariant) may be lost in the Flow Through/Wash fractions. Two modes ofoperation, namely the recycle chromatography mode and the continuouschromatography mode, provide improved recovery, while maintaining thetarget AR levels.

In one embodiment, in the recycle chromatography mode, the load materialis, in general, processed over multiple cycles. In implementing therecycle chromatography mode, the load material may be prepared such thatthe eluate contains the target product purity or AR level. Under theseconditions, the AR enriched material may be collected in the FlowThrough/wash fractions. This material may be pooled and additional freshload material is added to achieve the appropriate loading capacity forthe next cycle of chromatography on the same column. In particular, inone embodiment, the column is eluted under conditions where the boundproduct (having low AR levels) is recovered, and subsequentlyregenerated and equilibrated to prepare for the next cycle.

In the next cycle, the combined load (Flow Through/wash from cycle 1above, as well as fresh material) may be loaded to the target capacity.The Flow Through/wash fractions are collected and pooled. The column maybe then eluted to obtain the second eluate, again containing the targetlow AR composition. In one embodiment, this sequence is continued untilall the load materials are processed.

In another embodiment, by implementing the recycle chromatography mode,the material that would otherwise be discarded as AR enriched materialis further purified to “recover” pure protein product, thereby improvingthe overall recovery of the protein. In one embodiment, the level ofrecovery depends on the number of cycles employed.

For the recycle chromatography mode, several variations can be employed.In one embodiment, the entire pool of the Flow Through/wash fractionsare typically combined with fresh materials to maximize recovery of theentire operation. However, a portion of the flow through wash can bediscarded to achieve higher purity or efficiency. For example, in oneembodiment, certain fractions containing very high levels of AR speciescan be discarded. To enable such selective pooling, off-line, in-line orat line methods can be used to directly or indirectly measure the levelsof AR.

In another embodiment, the loading amount and the conditions forloading, washing and eluting can be modified for the second andsubsequent cycles to accommodate the higher levels of AR that will bepresent in the loading pool.

In still another embodiment, the last cycle of the method can beperformed under conditions such that the target purity and recovery canbe achieved to optimize overall recovery and purity.

A continuous chromatography mode provides additional advantages in termsof time efficiency. In one embodiment, in this mode of operation, two ormore columns are used. Specifically, as with the recycle mode, anappropriate condition for the load capacity, load, wash and elutionconditions are chosen for the operation. In one embodiment, the FlowThrough and wash fractions (or a portion thereof) may be directed to theload tank containing the fresh material. After completion of the loadand wash steps, the first column may be eluted and subsequentlyregenerated. Meanwhile, the second column may be loaded with thematerial that is a mix of fresh material and the wash and Flow Throughfrom the previous cycle. In one embodiment, the wash and Flow Throughfrom the second column may be again directed back to the load tank. Thesecond column may be then eluted and regenerated. In one embodiment, thefirst column is then ready to be loaded and the cycle continues. Thiscontinuous chromatography mode is efficient as the product is processedcontinuously and the purified product is obtained in a semi-continuousmanner.

Several variations of the continuous chromatography mode can beemployed. In one embodiment, the entire pool of the Flow Through/washfractions is combined with fresh materials to maximize recovery of theentire operation. However, a portion of the Flow Through wash can bediscarded to achieve higher purity or efficiency. For example, certainfractions containing very high levels of AR species can be discarded. Toenable such selective pooling, off-line, in-line or at line methods canbe used to measure directly or indirectly the levels of acidic species.

In another embodiment, the loading amount, conditions for loading,washing and eluting can be modified for the second and subsequent cyclesto accommodate the higher levels of AR that will be present in theloading pool. In still another embodiment, the last cycle of theoperation can be performed under different conditions to optimizeoverall recover and purity.

The recycle chromatography mode and the continuous chromatography modeare not limited to use with any particular chromatography resin. Themedia used for the recycle or continuous modes can be, for example, oneof many chromatographic resins with pendant hydrophobic and anionexchange functional groups, monolithic media, membrane adsorber media ordepth filtration media.

In certain embodiments, membrane depth filter-based media (“convectivemedia”) can be used with the recycle or continuous modes as theselectivity of separation is not required to be high given the fact thatthe less enriched portions of the product are “recycled” while the purefractions are selectively pooled.

Recycle chromatography mode and the continuous chromatography mode canbe used inconjunction with AEX, CEX, or MM chromatography methods, asdescribed herein, to produce the low AR compositions of the invention.For example, Example 11, below, describes the recycle mode ofchromatography for AR reduction using AEX, CEX, and MM technologies.

Hydrophobic Interaction Chromatography

The low AR compositions of the invention may also be prepared using ahydrophobic interaction chromatography (HIC) step in addition to thedisplacement chromatography step.

In performing the separation, the sample mixture is contacted with theHIC material, e.g., using a batch purification technique or using acolumn or membrane chromatography. Prior to HIC purification it may bedesirable to adjust the concentration of the salt buffer to achievedesired protein binding to the resin or the membrane.

Whereas ion exchange chromatography relies on the local charge of theprotein of interest for selective separation, hydrophobic interactionchromatography employs the hydrophobic properties of the proteins toachieve selective separation. Hydrophobic groups on the protein interactwith hydrophobic groups of the resin or the membrane. The morehydrophobic a protein is the stronger it will interact with the columnor the membrane. Thus the HIC step removes process-related impurities(e.g., HCPs) as well as product-related substances (e.g., aggregates andfragments).

Like ion exchange chromatography, a HIC column or membrane device canalso be operated in product a bind-elute mode, a flow-through, or ahybrid mode wherein the product exhibits binding to the chromatographicmaterial, yet can be washed from the column using a buffer that is thesame or substantially similar to the loading buffer. The details ofthese modes are outlined above in connection with AEX purification.As hydrophobic interactions are strongest at high ionic strength, thisform of separation is conveniently performed following salt elutionstep, such as those that are typically used in connection with ionexchange chromatography. Alternatively, salts can be added into a lowsalt level feed stream before this step. Adsorption of the antibody to aHIC column is favored by high salt concentrations, but the actualconcentrations can vary over a wide range depending on the nature of theprotein of interest, salt type and the particular HIC ligand chosen.Various ions can be arranged in a so-called soluphobic series dependingon whether they promote hydrophobic interactions (salting-out effects)or disrupt the structure of water (chaotropic effect) and lead to theweakening of the hydrophobic interaction. Cations are ranked in terms ofincreasing salting out effect as Ba2+; Ca2+; Mg2+; Li+; Cs+; Na+; K+;Rb+; NH4+, while anions may be ranked in terms of increasing chaotropiceffect as PO43−; SO42−; CH3CO3−; Cl−; Br−; NO3−; ClO4−; I−; SCN−.

In general, Na+, K+ or NH4+ sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship:(NH4)2SO4>Na2SO4>NaCl>NH4Cl>NaBr>NaSCN. In general, salt concentrationsof between about 0.75 M and about 2 M ammonium sulfate or between about1 and 4 M NaCl are useful.

HIC media normally comprise a base matrix (e.g., cross-linked agarose orsynthetic copolymer material) to which hydrophobic ligands (e.g., alkylor aryl groups) are coupled. A suitable HIC media comprises an agaroseresin or a membrane functionalized with phenyl groups (e.g., a PhenylSepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). ManyHIC resins are available commercially. Examples include, but are notlimited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or highsubstitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ HighPerformance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butylcolumns (Bio-Rad, California); WP HI-Propyl (C3)™ (J. T. Baker, N.J.);and Toyopearl™ ether, phenyl or butyl (TosoHaas, Pa.).

Viral Filtration

Viral filtration is a dedicated viral reduction step in the entirepurification process. This step is usually performed postchromatographic polishing steps. Viral reduction can be achieved via theuse of suitable filters including, but not limited to, Planova 20N™, 50N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMDMillipore, ViroS art CPV from Sartorius, or Ultipor DV20 or DV50™ filterfrom Pall Corporation. It will be apparent to one of ordinary skill inthe art to select a suitable filter to obtain desired filtrationperformance.

Ultrafiltration/Diafiltration

Certain embodiments of the present invention employ ultrafiltration anddiafiltration steps to further concentrate and formulate the protein ofinterest, e.g., an antibody product. Ultrafiltration is described indetail in: Microfiltration and Ultrafiltration: Principles andApplications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York,N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (TechnomicPublishing, 1986; ISBN No. 87762-456-9). One filtration process isTangential Flow Filtration as described in the Millipore catalogueentitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202(Bedford, Mass., 1995/96). Ultrafiltration is generally considered tomean filtration using filters with a pore size of smaller than 0.1 μm.By employing filters having such small pore size, the volume of thesample can be reduced through permeation of the sample buffer throughthe filter membrane pores while proteins, such as antibodies, areretained above the membrane surface.

Diafiltration is a method of using membrane filters to remove andexchange salts, sugars, and non-aqueous solvents, to separate free frombound species, to remove low molecular-weight species, and/or to causethe rapid change of ionic and/or pH environments. Microsolutes areremoved most efficiently by adding solvent to the solution beingdiafiltered at a rate approximately equal to the permeate flow rate.This washes away microspecies from the solution at a constant volume,effectively purifying the retained protein of interest. In certainembodiments of the present invention, a diafiltration step is employedto exchange the various buffers used in connection with the instantinvention, optionally prior to further chromatography or otherpurification steps, as well as to remove impurities from the proteinpreparations.

One of ordinary skill in the art can select appropriate membrane filterdevice for the UF/DF operation. Examples of membrane cassettes suitablefor the present invention include, but not limited to, Pellicon 2 orPellicon 3 cassettes with 10 kD, 30 kD or 50 kD membranes from EMDMillipore, Kvick 10 kD, 30 kD or 50 kD membrane cassettes from GEHealthcare, and Centramate or Centrasette 10 kD, 30 kD or 50 kDcassettes from Pall Corporation.

Exemplary Purification Strategies

In certain embodiments, primary recovery can proceed by sequentiallyemploying pH reduction, centrifugation, and filtration steps to removecells and cell debris (including HCPs) from the production bioreactorharvest. In certain embodiments, the present invention is directed tosubjecting a sample mixture from said primary recovery to one or moreAEX, CEX, and/or MM purification steps. Certain embodiments of thepresent invention will include further purification steps. Examples ofadditional purification procedures which can be performed prior to,during, or following the ion exchange chromatography method includeethanol precipitation, isoelectric focusing, reverse phase HPLC,chromatography on silica, chromatography on heparin Sepharose™, furtheranion exchange chromatography and/or further cation exchangechromatography, chromatofocusing, SDS-PAGE, ammonium sulfateprecipitation, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography (e.g., using protein G, anantibody, a specific substrate, ligand or antigen as the capturereagent).

Specific examples of such combinations of strategies is presented below,with specific data relating to particular combinations useful in thecontext of the instant invention included in Tables 80-87 and 76-78.

In certain embodiments the unbound Flow Through and wash fractions canbe further fractionated and a combination of fractions providing atarget product purity can be pooled.

In certain embodiments the protein concentration can be adjusted toachieve a differential partitioning behavior between the antibodyproduct and the product-related substances such that the purity and/oryield can be further improved. In certain embodiments the loading can beperformed at different protein concentrations during the loadingoperation to improve the product quality/yield of any particularpurification step.

In certain embodiments the column temperature can be independentlyvaried to improve the separation efficiency and/or yield of anyparticular purification step.

In certain embodiments, the loading and washing buffer matrices can bedifferent or composed of mixtures of chemicals, while achieving similar“resin interaction” behavior such that the above novel separation can beeffected. For example, but not by way of limitation, the loading andwashing buffers can be different, in terms of ionic strength or pH,while remaining substantially similar in function in terms of thewashout of the product achieved during the wash step. In certainembodiments, additives such as amino acids, sugars, PEG, etc can beadded to the load or wash steps to modulate the partitioning behavior toachieve the separation efficiency and/or yield.

In certain embodiments, the loading & washing steps can be controlled byin-line, at-line or off-line measurement of the product relatedimpurity/substance levels, either in the column effluent, or thecollected pool or both, so as to achieve the target product qualityand/or yield. In certain embodiments, the loading concentration can bedynamically controlled by in-line or batch or continuous dilutions withbuffers or other solutions to achieve the partitioning necessary toimprove the separation efficiency and/or yield.

V. Methods of Assaying Sample Purity

Assaying Host Cell Protein

The present invention also provides methods for determining the residuallevels of host cell protein (HCP) concentration in the low ARcompositions of the invention. As described above, HCPs are desirablyexcluded from the final target substance product. Exemplary HCPs includeproteins originating from the source of the antibody production. Failureto identify and sufficiently remove HCPs from the target antibody maylead to reduced efficacy and/or adverse reactions in a subject.

As used herein, the term “HCP ELISA” refers to an ELISA where the secondantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the antibody of interest. The secondantibody may be produced according to conventional methods known tothose of skill in the art. For example, the second antibody may beproduced using HCPs obtained by sham production and purification runs,i.e., the same cell line used to produce the antibody of interest isused, but the cell line is not transfected with antibody DNA. In anexemplary embodiment, the second antibody is produced using HCPs similarto those expressed in the cell expression system of choice, i.e., thecell expression system used to produce the target antibody.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, for example, but notlimited to goat anti-CHO, affinity purified (Cygnus). A labeled secondantibody, or blend of antibodies, specific to the HCPs produced from thecells used to generate the antibody, e.g., anti-CHO HCP Biotinylated, isadded, and binds to the HCPs within the sample. In certain embodimentsthe first and second antibodies are polyclonal antibodies. In certainaspects the first and second antibodies are blends of polyclonalantibodies raised against HCPs. The amount of HCP contained in thesample is determined using the appropriate test based on the label ofthe second antibody.

HCP ELISA may be used for determining the level of HCPs in an antibodycomposition, such as an eluate or flow-through obtained using theprocess described above. The present invention also provides acomposition comprising an antibody, wherein the composition has nodetectable level of HCPs as determined by an HCP Enzyme LinkedImmunosorbent Assay (“ELISA”).

Assaying Acidic Species (AR)

The levels of acidic species in the chromatographic samples producedusing the techniques described herein may be analyzed as described inthe Examples section. In certain embodiments a CEX-HPLC method isemployed. For example, but not by way of limitation, cation exchangechromatography can be performed on a Dionex ProPac WCX-10, Analyticalcolumn 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLC system canthen be used as the HPLC. In certain embodiments, mobile phases such as10 mM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B) can beused. In certain embodiments, a binary gradient (94% A, 6% B: 0-20 min;84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34min) can be used with detection at 280 nm. In certain embodiments,quantitation is based on the relative area percent of detected peaks. Incertain embodiments, the peaks that elute at relative residence timeless than a certain time are together represented as the acidic peaks.

Assaying Size Variants

In certain embodiments, the levels of aggregates, monomer, and fragmentsin the chromatographic samples produced using the techniques describedherein are analyzed. In certain embodiments, the aggregates, monomer,and fragments are measured using a size exclusion chromatographic (SEC)method for each molecule. For example, but not by way of limitation, aTSK-gel G3000SW×L, 5 μm, 125 Å, 7.8×300 mm column (Tosoh Bioscience) canbe used in connection with certain embodiments, while a TSK-gel SuperSW3000, 4 μm, 250 Å, 4.6×300 mm column (Tosoh Bioscience) can be used inalternative embodiments. In certain embodiments, the aforementionedcolumns are used along with an Agilent or a Shimazhu HPLC system. Incertain embodiments, sample injections are made under isocratic elutionconditions using a mobile phase consisting of, for example, 100 mMsodium sulfate and 100 mM sodium phosphate at pH 6.8, and detected withUV absorbance at 214 nm. In certain embodiments, the mobile phase willconsist of 1×PBS at pH 7.4, and elution profile detected with UVabsorbance at 280 nm. In certain embodiments, quantification is based onthe relative area of detected peaks.

Any additional technique, such as mass spectroscopy, can be used forassaying size variants.

VI. Methods of Treatment Using the Low AR Compositions of the Invention

The low AR compositions of the invention may be used to treat anydisorder in a subject for which the therapeutic protein comprised in thecomposition is appropriate for treating.

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the subject to thedisorder in question. In the case of an anti-TNFα antibody, or antigenbinding portion thereof, such as adalimumab, a therapeutically effectiveamount of the low AR composition may be administered to treat a disorderin which TNFα activity is detrimental.

A disorder in which TNFα activity is detrimental includes a disorder inwhich inhibition of TNFα activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFα in a biologicalfluid of a subject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody.

TNFα has been implicated in the pathophysiology of a wide variety of aTNFα-related disorders including sepsis, infections, autoimmunediseases, transplant rejection and graft-versus-host disease (see e.g.,Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024to Moeller et al.; European Patent Publication No. 260 610 B1 byMoeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452;Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).Accordingly, the low AR compositions or a low process-related impuritycompositions of the invention may be used to treat an autoimmunedisease, such as rheumatoid arthritis, juvenile idiopathic arthritis, orpsoriatic arthritis, an intestinal disorder, such as Crohn's disease orulcerative colitis, a spondyloarthropathy, such as ankylosingspondylitis, or a skin disorder, such as psoriasis.

Disorders in which TNFα activity is detrimental are well known in theart and described in detail in U.S. Pat. No. 8,231,876 and U.S. Pat. No.6,090,382, the entire contents of each of which are expresslyincorporated herein by reference. In one embodiment, “a disorder inwhich TNFα activity is detrimental” includes sepsis (including septicshock, endotoxic shock, gram negative sepsis and toxic shock syndrome),autoimmune diseases (including rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,multisystem autoimmune diseases, lupus (including systemic lupus, lupusnephritis and lupus cerebritis), Crohn's disease and autoimmune hearingloss), active axial spondyloarthritis (active axSpA) andnon-radiographic axial spondyloarthritis (nr-axSpA), infectious diseases(including malaria, meningitis, acquired immune deficiency syndrome(AIDS), influenza and cachexia secondary to infection), allograftrejection and graft versus host disease, malignancy, pulmonary disorders(including adult respiratory distress syndrome (ARDS), shock lung,chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis, silicosis, idiopathic interstitial lung disease and chronicobstructive airway disorders (COPD), such as asthma), intestinaldisorders (including inflammatory bowel disorders, idiopathicinflammatory bowel disease, Crohn's disease and Crohn's disease-relateddisorders (including fistulas in the bladder, vagina, and skin; bowelobstructions; abscesses; nutritional deficiencies; complications fromcorticosteroid use; inflammation of the joints; erythem nodosum;pyoderma gangrenosum; lesions of the eye, Crohn's related arthralgias,fistulizing Crohn's indeterminant colitis and pouchitis), cardiacdisorders (including ischemia of the heart, heart insufficiency,restenosis, congestive heart failure, coronary artery disease, anginapectoris, myocardial infarction, cardiovascular tissue damage caused bycardiac arrest, cardiovascular tissue damage caused by cardiac bypass,cardiogenic shock, and hypertension, atherosclerosis, cardiomyopathy,coronary artery spasm, coronary artery disease, valvular disease,arrhythmias, and cardiomyopathies), spondyloarthropathies (includingankylosing spondylitis, psoriatic arthritis/spondylitis, enteropathicarthritis, reactive arthritis or Reiter's syndrome, and undifferentiatedspondyloarthropathies), metabolic disorders (including obesity anddiabetes, including type 1 diabetes mellitus, type 2 diabetes mellitus,diabetic neuropathy, peripheral neuropathy, diabetic retinopathy,diabetic ulcerations, retinopathy ulcerations and diabeticmacrovasculopathy), anemia, pain (including acute and chronic pains,such as neuropathic pain and post-operative pain, chronic lower backpain, cluster headaches, herpes neuralgia, phantom limb pain, centralpain, dental pain, opioid-resistant pain, visceral pain, surgical pain,bone injury pain, pain during labor and delivery, pain resulting fromburns, including sunburn, post partum pain, migraine, angina pain, andgenitourinary tract-related pain including cystitis), hepatic disorders(including hepatitis, alcoholic hepatitis, viral hepatitis, alcoholiccirrhosis, a1 antitypsin deficiency, autoimmune cirrhosis, cryptogeniccirrhosis, fulminant hepatitis, hepatitis B and C, and steatohepatitis,cystic fibrosis, primary biliary cirrhosis, sclerosing cholangitis andbiliary obstruction), skin and nail disorders (including psoriasis(including chronic plaque psoriasis, guttate psoriasis, inversepsoriasis, pustular psoriasis and other psoriasis disorders), pemphigusvulgaris, scleroderma, atopic dermatitis (eczema), sarcoidosis, erythemanodosum, hidradenitis suppurative, lichen planus, Sweet's syndrome,scleroderma and vitiligo), vasculitides (including Behcet's disease),and other disorders, such as juvenile rheumatoid arthritis (JRA),endometriosis, prostatitis, choroidal neovascularization, sciatica,Sjogren's syndrome, uveitis, wet macular degeneration, osteoporosis andosteoarthritis.

As used herein, the term “subject” is intended to include livingorganisms, e.g., prokaryotes and eukaryotes. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In specificembodiments of the invention, the subject is a human.

As used herein, the term “treatment” or “treat” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder, as well asthose in which the disorder is to be prevented.

In one embodiment, the invention provides a method of administering alow AR composition comprising an anti-TNFα antibody, or antigen bindingportion thereof, to a subject such that TNFα activity is inhibited or adisorder in which TNFα activity is detrimental is treated. In oneembodiment, the TNFα is human TNFα and the subject is a human subject.In one embodiment, the anti-TNFα antibody is adalimumab, also referredto as HUMIRA®.

The low AR compositions can be administered by a variety of methodsknown in the art. Exemplary routes/modes of administration includesubcutaneous injection, intravenous injection or infusion. In certainaspects, a low AR compositions may be orally administered. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a low AR composition of theinvention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. With respectto low AR compositions comprising an anti-TNFα antibody, orantigen-binding portion thereof, such as adalimumab, an exemplary doseis 40 mg every other week. In some embodiments, in particular fortreatment of ulcerative colitis or Crohn's disease, an exemplary doseincludes an initial dose (Day 1) of 160 mg (e.g., four 40 mg injectionsin one day or two 40 mg injections per day for two consecutive days), asecond dose two weeks later of 80 mg, and a maintenance dose of 40 mgevery other week beginning two weeks later. Alternatively, for psoriasisfor example, a dosage can include an 80 mg initial dose followed by 40mg every other week starting one week after the initial dose.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

VII. Pharmaceutical Formulations Containing the Low AR Compositions ofthe Invention

The present invention further provides preparations and formulationscomprising the low AR compositions of the invention. It should beunderstood that any of the antibodies and antibody fragments describedherein, including antibodies and antibody fragments having any one ormore of the structural and functional features described in detailthroughout the application, may be formulated or prepared as describedbelow. When various formulations are described in this section asincluding an antibody, it is understood that such an antibody may be anantibody or an antibody fragment having any one or more of thecharacteristics of the antibodies and antibody fragments describedherein. In one embodiment, the antibody is an anti-TNFα antibody, orantigen-binding portion thereof.

In certain embodiments, the low AR compositions of the invention may beformulated with a pharmaceutically acceptable carrier as pharmaceutical(therapeutic) compositions, and may be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. The term “pharmaceutically acceptable carrier” meansone or more non-toxic materials that do not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. Such pharmaceutically acceptable preparations may also routinelycontain compatible solid or liquid fillers, diluents or encapsulatingsubstances which are suitable for administration into a human. The term“carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the antibodies of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The low AR compositions of the invention are present in a form known inthe art and acceptable for therapeutic uses. In one embodiment, aformulation of the low AR compositions of the invention is a liquidformulation. In another embodiment, a formulation of the low ARcompositions of the invention is a lyophilized formulation. In a furtherembodiment, a formulation of the low AR compositions of the invention isa reconstituted liquid formulation. In one embodiment, a formulation ofthe low AR compositions of the invention is a stable liquid formulation.In one embodiment, a liquid formulation of the low AR compositions ofthe invention is an aqueous formulation. In another embodiment, theliquid formulation is non-aqueous. In a specific embodiment, a liquidformulation of the low AR compositions of the invention is an aqueousformulation wherein the aqueous carrier is distilled water.

The formulations of the low AR compositions of the invention comprise anantibody in a concentration resulting in a w/v appropriate for a desireddose. The antibody may be present in the formulation at a concentrationof about 1 mg/ml to about 500 mg/ml, e.g., at a concentration of atleast 1 mg/ml, at least 5 mg/ml, at least 10 mg/ml, at least 15 mg/ml,at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35mg/ml, at least 40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, atleast 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml,at least 95 mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, atleast 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least 150mg/ml, at least 200 mg/ml, at least 250 mg/ml, or at least 300 mg/ml.

In a specific embodiment, a formulation of the low AR compositions ofthe invention comprises at least about 100 mg/ml, at least about 125mg/ml, at least 130 mg/ml, or at least about 150 mg/ml of an antibody ofthe invention.

In one embodiment, the concentration of antibody, which is included inthe formulation of the invention, is between about 1 mg/ml and about 25mg/ml, between about 1 mg/ml and about 200 mg/ml, between about 25 mg/mland about 200 mg/ml, between about 50 mg/ml and about 200 mg/ml, betweenabout 75 mg/ml and about 200 mg/ml, between about 100 mg/ml and about200 mg/ml, between about 125 mg/ml and about 200 mg/ml, between about150 mg/ml and about 200 mg/ml, between about 25 mg/ml and about 150mg/ml, between about 50 mg/ml and about 150 mg/ml, between about 75mg/ml and about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml,between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml andabout 125 mg/ml, between about 50 mg/ml and about 125 mg/ml, betweenabout 75 mg/ml and about 125 mg/ml, between about 100 mg/ml and about125 mg/ml, between about 25 mg/ml and about 100 mg/ml, between about 50mg/ml and about 100 mg/ml, between about 75 mg/ml and about 100 mg/ml,between about 25 mg/ml and about 75 mg/ml, between about 50 mg/ml andabout 75 mg/ml, or between about 25 mg/ml and about 50 mg/ml.

In a specific embodiment, a formulation of the low AR compositions ofthe invention comprises between about 90 mg/ml and about 110 mg/ml orbetween about 100 mg/ml and about 210 mg/ml of an antibody.

The formulations of the low AR compositions of the invention comprisingan antibody may further comprise one or more active compounds asnecessary for the particular indication being treated, typically thosewith complementary activities that do not adversely affect each other.Such additional active compounds are suitably present in combination inamounts that are effective for the purpose intended.

The formulations of the low AR compositions of the invention may beprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers, including, but not limited to buffering agents,saccharides, salts, surfactants, solubilizers, polyols, diluents,binders, stabilizers, salts, lipophilic solutions, amino acids,chelators, preservatives, or the like (Goodman and Gilman's ThePharmacological Basis of Therapeutics, 12^(th) edition, L. Brunton, etal. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1999)), in the form of lyophilized formulations or aqueous solutions ata desired final concentration. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as histidine, phosphate, citrate,glycine, acetate and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including trehalose, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactantssuch as TWEEN, polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine, oracetate. The saccharide excipient may be trehalose, sucrose, mannitol,maltose or raffinose. The surfactant may be polysorbate 20, polysorbate40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl₂,or CaCl₂

The formulations of the low AR compositions of the invention may includea buffering or pH adjusting agent to provide improved pH control. Aformulation of the invention may have a pH of between about 3.0 andabout 9.0, between about 4.0 and about 8.0, between about 5.0 and about8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5,between about 5.5 and about 8.0, between about 5.5 and about 7.0, orbetween about 5.5 and about 6.5. In a further embodiment, a formulationof the invention has a pH of about 3.0, about 3.5, about 4.0, about 4.5,about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2,about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In aspecific embodiment, a formulation of the invention has a pH of about6.0. One of skill in the art understands that the pH of a formulationgenerally should not be equal to the isoelectric point of the particularantibody to be used in the formulation.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the formulations of the invention as bufferingagents include, but are not limited to, glycine and histidine. Incertain embodiments, the buffering agent is chosen from histidine,citrate, phosphate, glycine, and acetate. In a specific embodiment, thebuffering agent is histidine. In another specific embodiment, thebuffering agent is citrate. In yet another specific embodiment, thebuffering agent is glycine. The purity of the buffering agent should beat least 98%, or at least 99%, or at least 99.5%. As used herein, theterm “purity” in the context of histidine and glycine refers to chemicalpurity of histidine or glycine as understood in the art, e.g., asdescribed in The Merck Index, 13^(th) ed., O'Neil et al. ed. (Merck &Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In one embodiment, the bufferingagent is at a concentration of about 1 mM, or of about 5 mM, or of about10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or ofabout 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, orof about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM,or of about 90 mM, or of about 100 mM. In one embodiment, the bufferingagent is at a concentration of 1 mM, or of 5 mM, or of 10 mM, or of 15mM, or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, orof 45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90mM, or of 100 mM. In a specific embodiment, the buffering agent is at aconcentration of between about 5 mM and about 50 mM. In another specificembodiment, the buffering agent is at a concentration of between 5 mMand 20 mM.

In certain embodiments, the formulation of the low AR compositions ofthe invention comprises histidine as a buffering agent. In oneembodiment the histidine is present in the formulation of the inventionat a concentration of at least about 1 mM, at least about 5 mM, at leastabout 10 mM, at least about 20 mM, at least about 30 mM, at least about40 mM, at least about 50 mM, at least about 75 mM, at least about 100mM, at least about 150 mM, or at least about 200 mM histidine. Inanother embodiment, a formulation of the invention comprises betweenabout 1 mM and about 200 mM, between about 1 mM and about 150 mM,between about 1 mM and about 100 mM, between about 1 mM and about 75 mM,between about 10 mM and about 200 mM, between about 10 mM and about 150mM, between about 10 mM and about 100 mM, between about 10 mM and about75 mM, between about 10 mM and about 50 mM, between about 10 mM andabout 40 mM, between about 10 mM and about 30 mM, between about 20 mMand about 75 mM, between about 20 mM and about 50 mM, between about 20mM and about 40 mM, or between about 20 mM and about 30 mM histidine. Ina further embodiment, the formulation comprises about 1 mM, about 5 mM,about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM,about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In aspecific embodiment, a formulation may comprise about 10 mM, about 25mM, or no histidine.

The formulations of the low AR compositions of the invention maycomprise a carbohydrate excipient. Carbohydrate excipients can act,e.g., as viscosity enhancing agents, stabilizers, bulking agents,solubilizing agents, and/or the like. Carbohydrate excipients aregenerally present at between about 1% to about 99% by weight or volume,e.g., between about 0.1% to about 20%, between about 0.1% to about 15%,between about 0.1% to about 5%, about 1% to about 20%, between about 5%to about 15%, between about 8% to about 10%, between about 10% and about15%, between about 15% and about 20%, between 0.1% to 20%, between 5% to15%, between 8% to 10%, between 10% and 15%, between 15% and 20%,between about 0.1% to about 5%, between about 5% to about 10%, orbetween about 15% to about 20%. In still other specific embodiments, thecarbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or at2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are chosen from, sucrose, trehalose, lactose, mannitol, andraffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98%, or at least 99%, or at least 99.5%.

In a specific embodiment, the formulations of the low AR compositions ofthe invention may comprise trehalose. In one embodiment, a formulationof the invention comprises at least about 1%, at least about 2%, atleast about 4%, at least about 8%, at least about 20%, at least about30%, or at least about 40% trehalose. In another embodiment, aformulation of the invention comprises between about 1% and about 40%,between about 1% and about 30%, between about 1% and about 20%, betweenabout 2% and about 40%, between about 2% and about 30%, between about 2%and about 20%, between about 4% and about 40%, between about 4% andabout 30%, or between about 4% and about 20% trehalose. In a furtherembodiment, a formulation of the invention comprises about 1%, about 2%,about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about40% trehalose. In a specific embodiment, a formulation of the inventioncomprises about 4%, about 6% or about 15% trehalose.

In certain embodiments, a formulation of the low AR compositions of theinvention comprises an excipient. In a specific embodiment, aformulation of the invention comprises at least one excipient chosenfrom: sugar, salt, surfactant, amino acid, polyol, chelating agent,emulsifier and preservative. In one embodiment, a formulation of theinvention comprises a salt, e.g., a salt selected from: NaCl, KCl,CaCl₂, and MgCl₂. In a specific embodiment, the formulation comprisesNaCl.

A formulation of the low AR compositions of the invention may compriseat least about 10 mM, at least about 25 mM, at least about 50 mM, atleast about 75 mM, at least about 80 mM, at least about 100 mM, at leastabout 125 mM, at least about 150 mM, at least about 175 mM, at leastabout 200 mM, or at least about 300 mM sodium chloride (NaCl). In afurther embodiment, the formulation may comprise between about 10 mM andabout 300 mM, between about 10 mM and about 200 mM, between about 10 mMand about 175 mM, between about 10 mM and about 150 mM, between about 25mM and about 300 mM, between about 25 mM and about 200 mM, between about25 mM and about 175 mM, between about 25 mM and about 150 mM, betweenabout 50 mM and about 300 mM, between about 50 mM and about 200 mM,between about 50 mM and about 175 mM, between about 50 mM and about 150mM, between about 75 mM and about 300 mM, between about 75 mM and about200 mM, between about 75 mM and about 175 mM, between about 75 mM andabout 150 mM, between about 100 mM and about 300 mM, between about 100mM and about 200 mM, between about 100 mM and about 175 mM, or betweenabout 100 mM and about 150 mM sodium chloride. In a further embodiment,the formulation may comprise about 10 mM, about 25 mM, about 50 mM,about 75 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM,about 175 mM, about 200 mM, or about 300 mM sodium chloride.

A formulation of the low AR compositions of the invention may alsocomprise an amino acid, e.g., lysine, arginine, glycine, histidine or anamino acid salt. The formulation may comprise at least about 1 mM, atleast about 10 mM, at least about 25 mM, at least about 50 mM, at leastabout 100 mM, at least about 150 mM, at least about 200 mM, at leastabout 250 mM, at least about 300 mM, at least about 350 mM, or at leastabout 400 mM of an amino acid. In another embodiment, the formulationmay comprise between about 1 mM and about 100 mM, between about 10 mMand about 150 mM, between about 25 mM and about 250 mM, between about 25mM and about 300 mM, between about 25 mM and about 350 mM, between about25 mM and about 400 mM, between about 50 mM and about 250 mM, betweenabout 50 mM and about 300 mM, between about 50 mM and about 350 mM,between about 50 mM and about 400 mM, between about 100 mM and about 250mM, between about 100 mM and about 300 mM, between about 100 mM andabout 400 mM, between about 150 mM and about 250 mM, between about 150mM and about 300 mM, or between about 150 mM and about 400 mM of anamino acid. In a further embodiment, a formulation of the inventioncomprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about400 mM of an amino acid.

The formulations of the low AR compositions of the invention may furthercomprise a surfactant. The term “surfactant” as used herein refers toorganic substances having amphipathic structures; namely, they arecomposed of groups of opposing solubility tendencies, typically anoil-soluble hydrocarbon chain and a water-soluble ionic group.Surfactants can be classified, depending on the charge of thesurface-active moiety, into anionic, cationic, and nonionic surfactants.Surfactants are often used as wetting, emulsifying, solubilizing, anddispersing agents for various pharmaceutical compositions andpreparations of biological materials. Pharmaceutically acceptablesurfactants like polysorbates (e.g., polysorbates 20 or 80); polyoxamers(e.g., poloxamer 188); Triton; sodium octyl glycoside; lauryl-,myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.), canoptionally be added to the formulations of the invention to reduceaggregation. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.Surfactants are particularly useful if a pump or plastic container isused to administer the formulation. The presence of a pharmaceuticallyacceptable surfactant mitigates the propensity for the protein toaggregate. The formulations may comprise a polysorbate which is at aconcentration ranging from between about 0.001% to about 1%, or about0.001% to about 0.1%, or about 0.01% to about 0.1%. In other specificembodiments, the formulations of the invention comprise a polysorbatewhich is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%,or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or0.015%, or 0.02%.

The formulations of the low AR compositions of the invention mayoptionally further comprise other common excipients and/or additivesincluding, but not limited to, diluents, binders, stabilizers,lipophilic solutions, preservatives, adjuvants, or the like.Pharmaceutically acceptable excipients and/or additives may be used inthe formulations of the invention. Commonly used excipients/additives,such as pharmaceutically acceptable chelators (for example, but notlimited to, EDTA, DTPA or EGTA) can optionally be added to theformulations of the invention to reduce aggregation. These additives areparticularly useful if a pump or plastic container is used to administerthe formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5%, or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield a microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005), and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofan antibody, as well known those in the art or as described herein.

In one embodiment, the low AR compositions of the invention areformulated with the same or similar excipients and buffers as arepresent in the commercial adalimumab (HUMIRA®) formulation, as describedin the “Highlights of Prescribing Information” for HUMIRA® (adalimumab)Injection (Revised January 2008) the contents of which are herebyincorporated herein by reference. For example, each prefilled syringe ofHUMIRA®, which is administered subcutaneously, delivers 0.8 mL (40 mg)of drug product to the subject. Each 0.8 mL of HUMIRA® contains 40 mgadalimumab, 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphatedihydrate, 1.22 mg dibasic sodium phosphate dihydrate, 0.24 mg sodiumcitrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol, 0.8 mgpolysorbate 80, and water for Injection, USP. Sodium hydroxide is addedas necessary to adjust pH.

It will be understood by one skilled in the art that the formulations ofthe low AR compositions of the invention may be isotonic with humanblood, wherein the formulations of the invention have essentially thesame osmotic pressure as human blood. Such isotonic formulations willgenerally have an osmotic pressure from about 250 mOSm to about 350mOSm. Isotonicity can be measured by, for example, using a vaporpressure or ice-freezing type osmometer. Tonicity of a formulation isadjusted by the use of tonicity modifiers. “Tonicity modifiers” arethose pharmaceutically acceptable inert substances that can be added tothe formulation to provide an isotonity of the formulation. Tonicitymodifiers suitable for this invention include, but are not limited to,saccharides, salts and amino acids.

In certain embodiments, the formulations of the low AR compositions ofthe invention have an osmotic pressure from about 100 mOSm to about 1200mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSmto about 800 mOSm, or from about 200 mOSm to about 600 mOSm, or fromabout 250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400mOSm, or from about 250 mOSm to about 350 mOSm.

The concentration of any one component or any combination of variouscomponents, of the formulations of the low AR compositions of theinvention is adjusted to achieve the desired tonicity of the finalformulation. For example, the ratio of the carbohydrate excipient toantibody may be adjusted according to methods known in the art (e.g.,U.S. Pat. No. 6,685,940). In certain embodiments, the molar ratio of thecarbohydrate excipient to antibody may be from about 100 moles to about1000 moles of carbohydrate excipient to about 1 mole of antibody, orfrom about 200 moles to about 6000 moles of carbohydrate excipient toabout 1 mole of antibody, or from about 100 moles to about 510 moles ofcarbohydrate excipient to about 1 mole of antibody, or from about 100moles to about 600 moles of carbohydrate excipient to about 1 mole ofantibody.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Pharmaceuticallyacceptable salts and those suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassium chloride, magnesium chloride,magnesium sulfate, and calcium chloride. In specific embodiments,formulations of the invention comprise NaCl, MgCl₂, and/or CaCl₂. In oneembodiment, concentration of NaCl is between about 75 mM and about 150mM. In another embodiment, concentration of MgCl₂ is between about 1 mMand about 100 mM. Pharmaceutically acceptable amino acids includingthose suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the low AR compositions of theinvention are pyrogen-free formulations which are substantially free ofendotoxins and/or related pyrogenic substances. Endotoxins includetoxins that are confined inside a microorganism and are released onlywhen the microorganisms are broken down or die. Pyrogenic substancesalso include fever-inducing, thermostable substances (glycoproteins)from the outer membrane of bacteria and other microorganisms. Both ofthese substances can cause fever, hypotension and shock if administeredto humans. Due to the potential harmful effects, even low amounts ofendotoxins must be removed from intravenously administeredpharmaceutical drug solutions. The Food & Drug Administration (“FDA”)has set an upper limit of 5 endotoxin units (EU) per dose per kilogrambody weight in a single one hour period for intravenous drugapplications (The United States Pharmacopeial Convention, PharmacopeialForum 26 (1):223 (2000)). When therapeutic proteins are administered inamounts of several hundred or thousand milligrams per kilogram bodyweight, as can be the case with antibodies, even trace amounts ofharmful and dangerous endotoxin must be removed. In certain specificembodiments, the endotoxin and pyrogen levels in the composition areless then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or lessthen 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.

When used for in vivo administration, the formulations of the low ARcompositions of the invention should be sterile. The formulations of theinvention may be sterilized by various sterilization methods, includingsterile filtration, radiation, etc. In one embodiment, the antibodyformulation is filter-sterilized with a presterilized 0.22-micronfilter. Sterile compositions for injection can be formulated accordingto conventional pharmaceutical practice as described in “Remington: TheScience & Practice of Pharmacy”, 21^(st) ed., Lippincott Williams &Wilkins, (2005). Formulations comprising antibodies, such as thosedisclosed herein, ordinarily will be stored in lyophilized form or insolution. It is contemplated that sterile compositions comprisingantibodies are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having an adapter thatallows retrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle. In one embodiment, a composition of theinvention is provided as a pre-filled syringe.

In one embodiment, a formulation of the low AR compositions of theinvention is a lyophilized formulation. The term “lyophilized” or“freeze-dried” includes a state of a substance that has been subjectedto a drying procedure such as lyophilization, where at least 50% ofmoisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Bulking agents known to theart include, for example, carbohydrates, including simple sugars such asdextrose, ribose, fructose and the like, alcohol sugars such asmannitol, inositol and sorbitol, disaccharides including trehalose,sucrose and lactose, naturally occurring polymers such as starch,dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serumalbumin), glycogen, and synthetic monomers and polymers.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest (such as an antibody of the invention), significantly preventsor reduces chemical and/or physical instability of the protein uponlyophilization and subsequent storage. Lyoprotectants include, but arenot limited to, sugars and their corresponding sugar alcohols; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher molecular weight sugar alcohols, e.g., glycerin,dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, andmannitol; propylene glycol; polyethylene glycol; PLURONICS™; andcombinations thereof. Additional examples of lyoprotectants include, butare not limited to, glycerin and gelatin, and the sugars mellibiose,melezitose, raffinose, mannotriose and stachyose. Examples of reducingsugars include, but are not limited to, glucose, maltose, lactose,maltulose, iso-maltulose and lactulose. Examples of non-reducing sugarsinclude, but are not limited to, non-reducing glycosides of polyhydroxycompounds selected from sugar alcohols and other straight chainpolyalcohols. Examples of sugar alcohols include, but are not limitedto, monoglycosides, compounds obtained by reduction of disaccharidessuch as lactose, maltose, lactulose and maltulose. The glycosidic sidegroup can be either glucosidic or galactosidic. Additional examples ofsugar alcohols include, but are not limited to, glucitol, maltitol,lactitol and iso-maltulose. In specific embodiments, trehalose orsucrose is used as a lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose)and antibody molecules of a formulation of the invention is at leastabout 10, at least about 50, at least about 100, at least about 200, orat least about 300. In another embodiment, the molar ratio of alyoprotectant (e.g., trehalose) and antibody molecules of a formulationof the invention is about 1, is about 2, is about 5, is about 10, about50, about 100, about 200, or about 300.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized antibody formulation in a diluent such that theantibody is dispersed in the reconstituted formulation. Thereconstituted formulation is suitable for administration (e.g.,parenteral administration) to a patient to be treated with the antibodyand, in certain embodiments of the invention, may be one which issuitable for intravenous administration.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. In some embodiments,diluents include, but are not limited to, sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In an alternative embodiment, diluents can includeaqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the low AR compositions of theinvention is a lyophilized formulation comprising an antibody of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of said antibodymay be recovered from a vial upon shaking said vial for 4 hours at aspeed of 400 shakes per minute wherein the vial is filled to half of itsvolume with the formulation. In another embodiment, a formulation of theinvention is a lyophilized formulation comprising an antibody of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of the antibody maybe recovered from a vial upon subjecting the formulation to threefreeze/thaw cycles wherein the vial is filled to half of its volume withsaid formulation. In a further embodiment, a formulation of theinvention is a lyophilized formulation comprising an antibody of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of the antibody maybe recovered by reconstituting a lyophilized cake generated from saidformulation.

In one embodiment, a reconstituted liquid formulation may comprise anantibody at the same concentration as the pre-lyophilized liquidformulation.

In another embodiment, a reconstituted liquid formulation may comprisean antibody at a higher concentration than the pre-lyophilized liquidformulation, e.g., .about 2 fold, about 3 fold, about 4 fold, about 5fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about10 fold higher concentration of an antibody than the pre-lyophilizedliquid formulation.

In yet another embodiment, a reconstituted liquid formulation maycomprise an antibody of the invention at a lower concentration than thepre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold or about 10 fold lower concentration of an antibody thanthe pre-lyophilized liquid formulation.

The pharmaceutical formulations of the low AR compositions of theinvention are typically stable formulations, e.g., stable at roomtemperature.

The terms “stability” and “stable” as used herein in the context of aformulation comprising an antibody of the invention refer to theresistance of the antibody in the formulation to aggregation,degradation or fragmentation under given manufacture, preparation,transportation and storage conditions. The “stable” formulations of theinvention retain biological activity under given manufacture,preparation, transportation and storage conditions. The stability of theantibody can be assessed by degrees of aggregation, degradation orfragmentation, as measured by HPSEC, static light scattering (SLS),Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD),urea unfolding techniques, intrinsic tryptophan fluorescence,differential scanning calorimetry, and/or ANS binding techniques,compared to a reference formulation. For example, a referenceformulation may be a reference standard frozen at −70° C. consisting of10 mg/ml of an antibody of the invention in PBS.

Therapeutic formulations of the low AR compositions of the invention maybe formulated for a particular dosage. Dosage regimens may be adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the antibody and theparticular therapeutic effect to be achieved, and (b) the limitationsinherent in the art of compounding such an antibody for the treatment ofsensitivity in individuals.

Therapeutic compositions of the low AR compositions of the invention canbe formulated for particular routes of administration, such as oral,nasal, pulmonary, topical (including buccal and sublingual), rectal,vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods known in the art of pharmacy. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will vary depending upon the subject being treated, and theparticular mode of administration. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the composition which produces atherapeutic effect. By way of example, in certain embodiments, theantibodies (including antibody fragments) are formulated for intravenousadministration. In certain other embodiments, the antibodies (includingantibody fragments) are formulated for local delivery to thecardiovascular system, for example, via catheter, stent, wire,intramyocardial delivery, intrapericardial delivery, or intraendocardialdelivery.

Formulations of the low AR compositions of the invention which aresuitable for topical or transdermal administration include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required (U.S. Pat. Nos. 7,378,110;7,258,873; 7,135,180; 7,923,029; and US Publication No. 20040042972).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the low AR compositions of the invention may be variedso as to obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without being toxic to thepatient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

In certain embodiments, antibodies of the invention can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention can cross the BBB (if desired),they can be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G.Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)Antimicrob. Agents Chemother. 39:180); surfactant Protein A receptor(Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species ofwhich may comprise the formulations of the invention, as well ascomponents of the invented molecules; p120 (Schreier et al. (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBSLett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.In one embodiment of the invention, the therapeutic compounds of theinvention are formulated in liposomes; in another embodiment, theliposomes include a targeting moiety. In another embodiment, thetherapeutic compounds in the liposomes are delivered by bolus injectionto a site proximal to the desired area. When administered in thismanner, the composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and may be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. Additionally oralternatively, the antibodies of the invention may be delivered locallyto the brain to mitigate the risk that the blood brain barrier slowseffective delivery.

In certain embodiments, the low AR compositions of the invention may beadministered with medical devices known in the art. For example, incertain embodiments an antibody or antibody fragment is administeredlocally via a catheter, stent, wire, or the like. For example, in oneembodiment, a therapeutic composition of the invention can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

The efficient dosages and the dosage regimens for the low ARcompositions of the invention depend on the disease or condition to betreated and can be determined by the persons skilled in the art. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

VIII. Alternative Formulations Containing the Low AR Compositions of theInvention

Alternative Aqueous Formulations

The invention also provides a low AR composition formulated as anaqueous formulation comprising a protein and water, as described in U.S.Pat. No. 8,420,081 and WO2012/065072, the contents of which are herebyincorporated by reference. In these aqueous formulations, the protein isstable without the need for additional agents. This aqueous formulationhas a number of advantages over conventional formulations in the art,including stability of the protein in water without the requirement foradditional excipients, increased concentrations of protein without theneed for additional excipients to maintain solubility of the protein,and low osmolality. These also have advantageous storage properties, asthe proteins in the formulation remain stable during storage, e.g.,stored as a liquid form for more than 3 months at 7° C. or freeze/thawconditions, even at high protein concentrations and repeated freeze/thawprocessing steps. In one embodiment, formulations described hereininclude high concentrations of proteins such that the aqueousformulation does not show significant opalescence, aggregation, orprecipitation.

In one embodiment, an aqueous low AR composition comprising a protein,e.g., an antibody, e.g., an anti-TNFα antibody or antigen biding portionthereof, and water is provided, wherein the formulation has certaincharacteristics, such as, but not limited to, low conductivity, e.g., aconductivity of less than about 2.5 mS/cm, a protein concentration of atleast about 10 μg/mL, an osmolality of no more than about 30 mOsmol/kg,and/or the protein has a molecular weight (Mw) greater than about 47kDa. In one embodiment, the formulation has improved stability, such as,but not limited to, stability in a liquid form for an extended time(e.g., at least about 3 months or at least about 12 months) or stabilitythrough at least one freeze/thaw cycle (if not more freeze/thaw cycles).In one embodiment, the formulation is stable for at least about 3 monthsin a form selected from the group consisting of frozen, lyophilized, orspray-dried.

In one embodiment, the formulation has a low conductivity, including,for example, a conductivity of less than about 2.5 mS/cm, a conductivityof less than about 2 mS/cm, a conductivity of less than about 1.5 mS/cm,a conductivity of less than about 1 mS/cm, or a conductivity of lessthan about 0.5 mS/cm.

In another embodiment, low AR compositions included in the formulationhave a given concentration, including, for example, a concentration ofat least about 1 mg/mL, at least about 10 mg/mL, at least about 50mg/mL, at least about 100 mg/mL, at least about 150 mg/mL, at leastabout 200 mg/mL, or greater than about 200 mg/mL. In another embodiment,the formulation of the invention has an osmolality of no more than about15 mOsmol/kg.

The aqueous formulations described herein do not rely on standardexcipients, e.g., a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyroprotectant, a basic component, and an acidic component. In otherembodiments of the invention, the formulation contains water, one ormore proteins, and no ionic excipients (e.g., salts, free amino acids).

In certain embodiments, the aqueous formulation as described hereincomprise a low AR composition comprising a protein concentration of atleast 50 mg/mL and water, wherein the formulation has an osmolality ofno more than 30 mOsmol/kg. Lower limits of osmolality of the aqueousformulation are also encompassed by the invention. In one embodiment theosmolality of the aqueous formulation is no more than 15 mOsmol/kg. Theaqueous formulation of the invention may have an osmolality of less than30 mOsmol/kg, and also have a high protein concentration, e.g., theconcentration of the protein is at least 100 mg/mL, and may be as muchas 200 mg/mL or greater. Ranges intermediate to the above recitedconcentrations and osmolality units are also intended to be part of thisinvention. In addition, ranges of values using a combination of any ofthe above recited values as upper and/or lower limits are intended to beincluded.

The concentration of the aqueous formulation as described herein is notlimited by the protein size and the formulation may include any sizerange of proteins. Included within the scope of the invention is anaqueous formulation comprising at least 40 mg/mL and as much as 200mg/mL or more of a protein, for example, 40 mg/mL, 65 mg/mL, 130 mg/mL,or 195 mg/ml, which may range in size from 5 kDa to 150 kDa or more. Inone embodiment, the protein in the formulation of the invention is atleast about 15 kD in size, at least about 20 kD in size; at least about47 kD in size; at least about 60 kD in size; at least about 80 kD insize; at least about 100 kD in size; at least about 120 kD in size; atleast about 140 kD in size; at least about 160 kD in size; or greaterthan about 160 kD in size. Ranges intermediate to the above recitedsizes are also intended to be part of this invention. In addition,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included.

The aqueous formulation as described herein may be characterized by thehydrodynamic diameter (D_(h)) of the proteins in solution. Thehydrodynamic diameter of the protein in solution may be measured usingdynamic light scattering (DLS), which is an established analyticalmethod for determining the D_(h) of proteins. Typical values formonoclonal antibodies, e.g., IgG, are about 10 nm. Low-ionicformulations may be characterized in that the D_(h) of the proteins arenotably lower than protein formulations comprising ionic excipients. Ithas been discovered that the D_(h) values of antibodies in aqueousformulations made using the disfiltration/ultrafilteration (DF/UF)process, as described in U.S. Pat. No. 8,420,081, using pure water as anexchange medium, are notably lower than the D_(h) of antibodies inconventional formulations independent of protein concentration. In oneembodiment, antibodies in the aqueous formulation as described hereinhave a D_(h) of less than 4 nm, or less than 3 nm.

In one embodiment, the D_(h) of the protein in the aqueous formulationis smaller relative to the D_(h) of the same protein in a bufferedsolution, irrespective of protein concentration. Thus, in certainembodiments, protein in an aqueous formulation made in accordance withthe methods described herein, will have a D_(h) which is at least 25%less than the D_(h) of the protein in a buffered solution at the samegiven concentration. Examples of buffered solutions include, but are notlimited to phosphate buffered saline (PBS). In certain embodiments,proteins in the aqueous formulation of the invention have a D_(h) thatis at least 50% less than the D_(h) of the protein in PBS in at thegiven concentration; at least 60% less than the D_(h) of the protein inPBS at the given concentration; at least 70% less than the D_(h) of theprotein in PBS at the given concentration; or more than 70% less thanthe D_(h) of the protein in PBS at the given concentration. Rangesintermediate to the above recited percentages are also intended to bepart of this invention, e.g., about 55%, 56%, 57%, 64%, 68%, and soforth. In addition, ranges of values using a combination of any of theabove recited values as upper and/or lower limits are intended to beincluded, e.g., about 50% to about 80%.

In one aspect, the aqueous formulation includes the protein at a dosageof about 0.01 mg/kg-10 mg/kg. In another aspect, the dosages of theprotein include approximately 1 mg/kg administered every other week, orapproximately 0.3 mg/kg administered weekly. A skilled practitioner canascertain the proper dosage and regime for administering to a subject.

Alternative Solid Unit Formulations

The invention also provides a low AR composition of the inventionformulated asa stable solid composition of a protein (preferably atherapeutic protein) and a stabilizer, referred to herein as solidunits, as described in Attorney Docket No. 117813-31001 (U.S.Provisional Patent Application 61/893,123), the contents of which arehereby incorporated by reference herein. Specifically, it has beendiscovered that despite having a high proportion of sugar relative tothe protein, the solid units of the invention maintain structuralrigidity and resist changes in shape and/or volume when stored underambient conditions, e.g., room temperature and humidity, for extendedperiods of time. The solid units of the invention remain free-flowingand are able to maintain long-term physical and chemical stability ofthe protein without significant degradation and/or aggregate formation.The solid units of the invention have many advantages over the art,including that they can be formulated for oral delivery and are easilyreconstituted in a diluent, such as water. Because the solid units arereadily dissolved, they may be used in dual chamber delivery devices andmay be prepared directly in a device for patient use.

As used herein, the term “solid unit,” refers to a composition which issuitable for pharmaceutical administration and comprises a protein,e.g., an antibody or peptide, and a stabilizer, e.g., a sugar. The solidunit has a structural rigidity and resistance to changes in shape and/orvolume. In a preferred embodiment, the solid unit is obtained bylyophilizing a pharmaceutical formulation of a therapeutic protein. Thesolid unit may be any shape, e.g., geometric shape, including, but notlimited to, a sphere, a cube, a pyramid, a hemisphere, a cylinder, ateardrop, and so forth, including irregularly shaped units. In oneembodiment, the solid unit has a volume ranging from about 1 μl to about20 μl. In one embodiment, the solid unit is not obtained using spraydrying techniques, e.g., the solid unit is not a powder or granule. Asused herein, the phrase “a plurality of solid units” refers to acollection or population of solid units, wherein the collectioncomprises two or more solid units having a substantially uniform shape,e.g., sphere, and/or volume distribution. In one embodiment, theplurality of solid units is free-flowing.

IX. Kits and Articles of Manufacture Comprising the Low AR Compositionsof the Invention

Also within the scope of the present invention are kits comprising thelow AR compositions of the invention and instructions for use. The term“kit” as used herein refers to a packaged product comprising componentswith which to administer the antibody, or antigen-binding portionthereof, of the invention for treatment of a disease or disorder. Thekit may comprise a box or container that holds the components of thekit. The box or container is affixed with a label or a Food and DrugAdministration approved protocol. The box or container holds componentsof the invention which may be contained within plastic, polyethylene,polypropylene, ethylene, or propylene vessels. The vessels can becapped-tubes or bottles. The kit can also include instructions foradministering an antibody of the invention.

The kit can further contain one more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent orone or more additional antibodies of the invention (e.g., an antibodyhaving a complementary activity which binds to an epitope in the TNFαantigen distinct from a first anti-TNFα antibody). Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a liquid formulation or lyophilizedformulation of an antibody or antibody fragment thereof of theinvention. In one embodiment, a container filled with a liquidformulation of the invention is a pre-filled syringe. In a specificembodiment, the formulations of the invention are formulated in singledose vials as a sterile liquid. For example, the formulations may besupplied in 3 cc USP Type I borosilicate amber vials (WestPharmaceutical Services—Part No. 6800-0675) with a target volume of 1.2mL. Optionally associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In one embodiment, a container filled with a liquid formulation of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a liquid formulationof the invention. Pre-filled syringes that may be used are described in,for example, but not limited to, PCT Publications WO05032627,WO08094984, WO9945985, WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat.No. 5,607,400, U.S. Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S.Pat. No. 7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797,U.S. Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S. Pat. No.7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120, U.S. Pat.No. 7,645,267, and US Patent Publication No. US20050075611. Pre-filledsyringes may be made of various materials. In one embodiment apre-filled syringe is a glass syringe. In another embodiment apre-filled syringe is a plastic syringe. One of skill in the artunderstands that the nature and/or quality of the materials used formanufacturing the syringe may influence the stability of a proteinformulation stored in the syringe. For example, it is understood thatsilicon based lubricants deposited on the inside surface of the syringechamber may affect particle formation in the protein formulation. In oneembodiment, a pre-filled syringe comprises a silicone based lubricant.In one embodiment, a pre-filled syringe comprises baked on silicone. Inanother embodiment, a pre-filled syringe is free from silicone basedlubricants. One of skill in the art also understands that small amountsof contaminating elements leaching into the formulation from the syringebarrel, syringe tip cap, plunger or stopper may also influence stabilityof the formulation. For example, it is understood that tungstenintroduced during the manufacturing process may adversely affectformulation stability. In one embodiment, a pre-filled syringe maycomprise tungsten at a level above 500 ppb. In another embodiment, apre-filled syringe is a low tungsten syringe. In another embodiment, apre-filled syringe may comprise tungsten at a level between about 500ppb and about 10 ppb, between about 400 ppb and about 10 ppb, betweenabout 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb,between about 100 ppb and about 10 ppb, between about 50 ppb and about10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising antibodies of the invention arealso provided that are useful for various purposes, e.g., research anddiagnostic including for purification or immunoprecipitation of proteinof interest from cells, detection of the protein of interest in vitro orin vivo. For isolation and purification of a protein of interest, thekit may contain an antibody coupled to beads (e.g., sepharose beads).Kits may be provided which contain the antibodies for detection andquantitation of a protein of interest in vitro, e.g., in an ELISA or aWestern blot. As with the article of manufacture, the kit comprises acontainer and a label or package insert on or associated with thecontainer. The container holds a composition comprising at least oneantibody of the invention. Additional containers may be included thatcontain, e.g., diluents and buffers, control antibodies. The label orpackage insert may provide a description of the composition as well asinstructions for the intended in vitro or diagnostic use.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free solution comprising an antibody that issuitable for parenteral administration. In another embodiment, the unitdosage form is provided as a sterile lyophilized powder comprising anantibody that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses sterile solutions suitable for each deliveryroute. The invention further encompasses sterile lyophilized powdersthat are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of apharmaceutical agent contained within said packaging material, whereinsaid pharmaceutical agent comprises a liquid formulation containing anantibody. The packaging material includes instruction means whichindicate how that said antibody can be used to prevent, treat and/ormanage one or more symptoms associated with a disease or disorder.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way.

X. EXAMPLES

Example 1 Method for Reducing the Extent of Acidic Species in CellCulture by the Addition of Medium Components

Production of recombinant proteins by host cells can result inproduct-related charge heterogeneities present in the population ofproteins produced by the cells. The presence of acidic species in thepopulation of proteins is an example of a product-related chargeheterogeneity. Control of the amount of acidic species present in thepopulation of proteins produced by the host cells can be accomplished bymodifying the culture conditions of the host cells.

The experiments in this Example demonstrate that supplementation of cellculture medium with supplemental amounts of amino acids, calciumchloride and niacinamide enhances product quality by decreasing theamount of acidic species in the culture harvest. The amino acidsincluded in the study were arginine, lysine, ornithine and histidine,which belong to the group of amino acids that are basic. The studyincludes examples from multiple cell lines and antibodies, in shakeflasks and bioreactors and in batch and fed-batch culture formats. Adose dependent effect in the extent of reduction of acidic species withincreasing concentrations of the supplements was observed. In addition,the possibility to supplement these medium additives individually or insuitable combinations for acidic species reduction was alsodemonstrated.

Materials and Methods

Cell Source and Adaptation Cultures

Three adalimumab producing cell lines (cell line 1, cell line 2, andcell line 3), one mAb1 producing cell line and one mAb2 producing cellline were employed in the studies covered below. For adalimumabproducing cell lines, cells were cultured in their respective growthmedia (chemically defined media (media 1) or a hydrolysate based media(media 2 or media 3)) in a combination of vented non-baffled shakeflasks (Corning) on a shaker platform at 110 RPM (cell line 1), 180 RPM(cell line 2), 140 RPM (cell line 3) and 10 L or 20 L wave bags (GE).For experiments with cells in the hydrolysate based media (media 3),cells were thawed in media 1 and then adapted to media 3 over a fewpassages. Cultures were propagated in a 35° C., 5% CO₂ incubator forcell line 1 and 2 and in a 36° C., 5% CO₂ incubator for cell line 3 inorder to obtain the required number of cells to be able to initiateproduction stage cultures.

For the mAb1 producing cell line, cells were cultured in chemicallydefined growth media (media 1) in a combination of vented non-baffledshake flasks (Corning) on a shaker platform at 130 RPM and 20 L wavebags (GE). Cultures were propagated in a 36° C., 5% CO₂ incubator toobtain the required number of cells to be able to initiate productionstage cultures.

For the mAb2 producing cell line, cells were cultured in chemicallydefined growth media (media 1) in a combination of vented non-baffledshake flasks (Corning) on a shaker platform at 140 RPM and 20 L wavebags (GE). Cultures were propagated in a 35° C., 5% CO₂ incubator toobtain the required number of cells to be able to initiate productionstage cultures.

Cell Culture Media

Growth and production media were prepared from either a chemicallydefined media formulation (media 1) or hydrolysate-based mediumformulations (media 2 and media 3). For preparation of the media 1, themedia (IVGN GIA-1, a proprietary basal media formulation fromInvitrogen) was supplemented with L-glutamine, sodium bicarbonate,sodium chloride, and methotrexate solution. Production media consistedof all the components in the growth medium, excluding methotrexate. Forcell line 1, both growth and production medium were also supplementedwith insulin. For mAb1 and mAb2 producing cell lines, the growth mediumwere also supplemented with insulin.

For the hydrolysate-based formulation (media 2), the growth media wascomposed of PFCHO (proprietary chemically defined formulation fromSAFC), Dextrose, L-Glutamine, L-Asparagine, HEPES, Poloxamer 188, FerricCitrate, Recombinant Human Insulin, Yeastolate (BD), Phytone Peptone(BD), Mono- and Di-basic Sodium Phosphate, Sodium Bicarbonate, SodiumChloride and methotrexate. Production media consisted of all thecomponents listed in the growth medium, excluding methotrexate.

For the hydrolysate-based formulation (media 3), the growth media wascomposed of OptiCHO (Invitrogen), L-Glutamine, Yeastolate (BD), PhytonePeptone (BD) and methotrexate. Production media consisted of all thecomponents listed in the growth medium, excluding methotrexate.

Amino acids used for the experiments were reconstituted in Milli-Q waterto make a 100 g/L stock solution, which was subsequently supplemented toboth growth and production basal media. After addition of amino acids,media was brought to a pH similar to unsupplemented (control) mediausing 5N hydrochloric acid/5N NaOH, and it was brought to an osmolalitysimilar to unsupplemented (control) media by adjusting the concentrationof sodium chloride.

Calcium Chloride Dihydrate (Sigma or Fluka) used for the experimentswere reconstituted in Milli-Q water to make a stock solution, which wassubsequently supplemented to the production basal media. After additionof calcium chloride, media was brought to a pH similar tonon-supplemented (control) media using 6N hydrochloric acid/5N NaOH, andit was brought to an osmolality similar to non-supplemented (control)media by adjusting the concentration of sodium chloride.

Niacinamide (Sigma or Calbiochem) used for the experiments werereconstituted in Milli-Q water to make a stock solution, which wassubsequently supplemented to the production basal media. After additionof niacinamide, media was brought to a pH similar to non-supplemented(control) media using 6N hydrochloric acid/5N NaOH, and it was broughtto an osmolality similar to non-supplemented (control) media byadjusting the concentration of sodium chloride.

All media was filtered through Corning 1 L filter systems (0.22 μm PES)and stored at 4° C. until usage.

TABLE 3 List of medium additives supplemented to culture media CatalogNo./Source Medium of medium additive supplements Arginine Sigma, A8094Lysine Calbiochem, 4400 Histidine Sigma, H5659 Ornithine Sigma, 06503Calcium Fulka, 21097 Chloride Sigma, C8106 Niacinamide Calbiochem,481907 Sigma, N0636

Production Cultures

Production cultures were initiated either in 500 ml shake flasks(Corning) or in 3 L Bioreactors (Applikon). For shake flask experiments,duplicate 500 mL Corning vented non-baffled shake flasks (200 mL workingvolume) were used for each condition. The shake flasks were kept inincubators either maintained at 35° C. or 36° C. and 5% CO₂ on shakerplatforms that were either set at 110 rpm for adalimumab producing cellline 1, 180 rpm for adalimumab producing cell line 2, 140 rpm foradalimumab producing cell line 3, for 130 rpm for mAb1 producing cellline, or 140 rpm for mAb2 producing cell line. For the bioreactorexperiments, 3 L bioreactors (1.5 L working volume) were run at 35° C.,30% dissolved oxygen (DO), 200 rpm, pH profile from 7.1 to 6.9 in threedays and pH 6.9 thereafter. In all experiments, the cells weretransferred from the seed train to the production stage at a split ratioof 1:5.

Cultures were run in either batch or fed-batch mode. In the batch mode,cells were cultured in the respective production medium. 1.25% (v/v) of40% glucose stock solution was fed when the media glucose concentrationreduced to less than 3 g/L. In the fed-batch mode, cultures were runwith either the IVGN feed (proprietary chemically defined feedformulation from Invitrogen) as per the following feed schedule—(4%(v/v)—day 6, day 7, and day 8, respectively) along with 10× Ex-CellPFCHO feed (proprietary chemically defined formulation)—3% (v/v) on day3. The cultures were also fed with 1.25% (v/v) of 40% glucose stocksolution when the glucose concentration was below 3.0 g/L.

Retention samples for titer analysis, of 2×1.5 mL, were collected dailyfor the bioreactor experiments beginning on Day 8, and frozen at −80° C.The samples taken from each were later submitted for titer analysis.

The harvest procedure of the shake flasks and reactors involvedcentrifugation of the culture sample at 3,000 RPM for 30 min and storageof supernatant in PETG bottles at −80° C. before submission for ProteinA purification and WCX-10 analysis.

WCX-10 Assay

This method is employed towards the quantification of the acidic speciesand other variants present in cell culture harvest samples. Cationexchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column (Dionex, Calif.).

For adalimumab and mAb1 samples, the mobile phases used were 10 mMSodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). Abinary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A,100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at280 nm.

For mAb2 samples, the mobile phases used were 20 mM(4-Morpholino)ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobile phaseA) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B). Anoptimized gradient (minute/% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3,58/3 was used with detection at 280 nm.

Quantitation is based on the relative area percent of detected peaks.The peaks that elute at relative residence time earlier than the mainpeak corresponding to the drug product are together represented as theacidic peaks (FIG. 1).

Lysine-C Peptide Mapping for Methylglyoxal (MGO) Quantification

Typical trypsin digestion employed almost universally for peptidemapping cleaves a denatured, reduced and alkylated protein at thecarboxyl side of the two basic amino acids, lysine and arginine.Methylglyoxal (MGO) is a small molecule metabolite derived as aglycolysis byproduct which can modify arginine residues. A modificationof an arginine prevents trypsin from cutting this site and results in amis-cleavage. The challenge of quantifying the amount of MGO modifiedpeptide is that it is not compared to an equivalent non-modified peptidebut rather two parental cleaved peptides which will likely havedifferent ionization potential than the modified peptide. In order todetermine a truly accurate direct measurement of an MGO-modifiedpeptide, it must be compared to its non-modified counterpart andexpressed as a percent. Using endoproteinase lysine-C as an alternativeenzyme, cleavages only occur at lysine residues. The result is a directcomparison of the same peptide with and without an MGO modificationwhich provides a high degree of accuracy in quantifying even tracelevels of the modified species.

Procedure: Samples were diluted to a nominal concentration of 4 mg/mL. 8M guanidine-HCl was added to the sample in a 3:1 ratio resulting in a 1mg/mL concentration in 6M guanidine-HCl. The samples were reduced with10 mM final conc. DTT for 30 minutes at 37° C. followed by an alkylationwith 25 mM final concentration iodoacetic acid for 30 minutes at 37° C.in the dark. The samples were then buffer exchanged into 10 mM Tris pH8.0 using NAP-5 columns. The samples were then digested for 4 hours at37° C. using endoproteinase Lys-C at an enzyme to protein ratio of 1:20.The digest was quenched by adding 5 μL of formic acid to each sample.Samples ere analyzed by LC/MS peptide mapping. Briefly, 50 μL of samplewas loaded onto a Waters BEH C18 1.7μ 1.0×150 mm UPLC column with 98%0.08% formic acid, 0.02% TFA in water and 2% 0.08% formic acid, 0.02%TFA in acetonitrile. The composition was changed to 65% 0.08% formicacid, 0.02% TFA in water and 35% 0.08% formic acid, 0.02% TFA inacetonitrile in 135 minutes using a Waters Acquity UPLC system. Elutingpeaks were monitored using a Thermo Scientific LTQ-Orbitrap MassSpectrometer. Specific mass traces were extracted for both modified andnon-modified peptides in order to accurately quantify the total amountof MGO modification at each site. Mass spectra were also analyzed forthe specific region of the chromatogram to confirm the peptide identity.An example data set is shown in FIG. 162.

Results

Effect of Arginine Supplementation to Cell Culture Media

The addition of arginine was tested in several experimental systemscovering multiple cell lines, media and monoclonal antibodies. Thefollowing is a detailed description of two representative experimentswhere two different adalimumab producing cell lines (cell line 2 andcell line 3) were cultured in a chemically defined media (media 1).

Cell line 2 was cultured in media 1 with different total amounts ofarginine (1 (control), 1.25, 1.5, 2, 3, 5, 9 g/L). The cultures wereperformed in shake flasks in batch format with only glucose feed asdescribed in the materials and methods. The cells grew to maximum viablecell densities (VCD) in the range of 18-22×10⁶ cells/ml for thedifferent conditions tested. The growth and viability profiles werecomparable between the different test conditions, although a slightdecrease in viable cell density profile was observed in samples with the9 g/L arginine test condition (FIGS. 1 and 2). The harvest titers werecomparable between the conditions (FIG. 3). On Day 10 and Day 12 ofculture, duplicate shake flasks for each of the conditions wereharvested and then subsequently analyzed using WCX-10 post Protein Apurification and the percentages of total peak(s) area corresponding tothe acidic species were quantified (FIGS. 4 and 5). The percentage ofacidic species in the control sample was as high as 19.7% on day 10. Inthe sample with the highest total concentration of arginine in thisexperiment (9 g/L), the percentage of acidic species was reduced to12.2%. A dose dependent decrease in acidic species was observed in testconditions with arginine concentrations beyond 2 g/L (FIG. 4). A similartrend in reduction of acidic species with arginine increase was alsoobserved in the day 12 harvest samples (FIG. 5). Further, while theextent of acidic species in the 1 g/L arginine samples increased from19.7% (day 10 harvest) to 25.5% (day 12 harvest), this increase in the 9g/L arginine test condition was significantly smaller from 12.2% (day 10harvest) to 13.9% (day 12 harvest). Thus, the increase of total arginineled to a reduction in the extent of total acidic species at a particulartime point in culture as well the rate of increase of acidic specieswith time of culture.

Cell line 3 was cultured in media 1 with different total amounts ofarginine (1 (control), 3, 5, 7, 9 g/L). The cultures were performed inshake flasks in batch format with only glucose feed as described in thematerials and methods. The cells grew to maximum VCD in the range of7-10×10⁶ cells/ml for the different conditions tested. The growth andviability profiles were comparable between the different testconditions, although a slight decrease in viable cell density andviability profiles was observed in samples with the 9 g/L argininecondition (FIGS. 6 and 7). The product titer was also comparable betweenall conditions (FIG. 8). On Day 10 of culture, duplicate shake flasksfor each of the conditions were harvested and then subsequently analyzedusing WCX-10 post Protein A purification and the percentages of totalpeak(s) area corresponding to the acidic species were quantified (FIG.9). The percentage of acidic species in the control sample was as highas 23.3% on day 10. In the sample with the highest total concentrationof arginine in this experiment (9 g/L), the percentage of acidic specieswas reduced to 17.0%. A dose dependent decrease in acidic species wasobserved in conditions with higher concentrations of arginine.

Additional experiments were performed with multiple cell lines inchemically defined or hydrolysate based media to demonstrate the widerange of applicability of this method. The experimental setup for eachof these experiments was similar to that described above. The summariesof results of the different experiments performed for adalimumab aresummarized in FIGS. 10, 11, and 12. A reduction in acidic species withincreased arginine concentration was also observed in each case.

In addition to adalimumab, the utility of this method for acidic speciesreduction was also demonstrated for processes involving two other mAbproducing cell lines (cell lines producing mAb1 and mAb2). Theexperimental setup for each of these experiments was similar to thatdescribed in section above and in the materials and methods. Thereduction of acidic species with increased arginine concentration forexperiments corresponding to each mAb is summarized in FIGS. 13 and 14.For mAb2, a significant reduction in acidic species was observed atarginine concentration of 9 g/L.

In U.S. Application Ser. No. 61/893,088, (the contents of which areincorporated herein by reference), we describe the utility of argininesupplementation to culture media towards modulation of the lysinevariant distribution. It is possible that a fraction of acidic speciesalso shifted along with shift in lysine variants (from Lys 0 to Lys1 andLys2), in addition to the fraction of acidic species that is completelyremoved from the entire protein population. To estimate the acidicspecies reduction that is independent of this redistribution of lysinevariants, Protein A eluate samples from a representative set of argininesupplementation experiments were pre-treated with the enzymecarboxypeptidase before WCX-10. One set of samples from adalimumabexperiment and another set of samples from a mAb2 experiment were usedfor this analysis. The carboxypeptidase treatment of the samplesresulted in the cleavage of the C-terminal lysine residues asdemonstrated by the complete conversion of Lys1/Lys2 to Lys 0 in each ofthese samples (data not shown here). As a result of this conversion, theacidic species quantified in these samples corresponded to an aggregatesum of acidic species that would be expected to also include thosespecies that may have previously shifted corresponding to the lysinevariant shift and perhaps gone unaccounted for in the samples that werenot treated with carboxypeptidase prior to WCX-10. A dose dependentreduction in acidic species was observed in the carboxypeptidase treatedsamples with increasing concentration arginine (FIGS. 15 and 16). Thissuggests that the acidic species reduction described here is notcompletely attributed to a probable shift of the acidic speciescorresponding to the lysine variant redistribution.

Effect of Lysine Supplementation to Cell Culture Media

The addition of lysine was tested in several experimental systemscovering multiple cell lines, media and monoclonal antibodies. Thefollowing is a detailed description of two representative experimentswhere two different cell lines (cell line 2 and cell line 3) werecultured in a chemically defined media (media 1) for the production ofadalimumab.

Cell line 2 was cultured in media 1 with different total concentrationsof lysine (1 (control), 5, 7, 9, 11 g/L). The cultures were performed inshake flasks in batch format with only glucose feed as described in thematerials and methods. The cells grew to maximum viable cell densities(VCD) in the range of 17-23×10⁶ cells/ml for the different conditionstested. A slight dose dependent decrease in viable cell density profilewas observed in all samples with respect to the control sample (FIG.17). The viability profiles were comparable between the conditions (FIG.18). On Days 10 and 11 of culture samples were collected for titeranalysis (FIG. 19). The titers for all conditions were comparable. OnDay 11 of culture, duplicate shake flasks for each of the conditionswere harvested and then subsequently analyzed using WCX-10 post ProteinA purification and the percentages of total peak(s) area correspondingto the acidic species were quantified (FIG. 20). The percentage ofacidic species in the control was as high as 26.5%. In the sample withthe highest tested concentration of lysine in this experiment (11 g/L),the percentage of acidic species was reduced to 15.0%. A dose dependentdecrease in acidic species was observed in test conditions with highertotal concentrations of lysine.

Cell line 3 was cultured in media 1 with different total concentrationsof lysine (1 (control), 3, 5, 7, 9, 11 g/L). The cultures were performedin shake flasks in batch format with only glucose feed as described inthe materials and methods. The cells grew to maximum VCD in the range of9.5-11.5×10⁶ cells/ml for the different conditions tested. The growthand viability profiles were comparable between the different testconditions, although a slight decrease in viable cell density andviability profiles was observed in samples with higher lysineconcentrations than that in the control sample (FIGS. 21 and 22). OnDays 10, 11 and 12 of culture samples were collected for titer analysis(FIG. 23). The titers for all conditions were comparable. On Day 12 ofculture, duplicate shake flasks for each of the conditions wereharvested and then subsequently analyzed using WCX-10 post Protein Apurification and the percentages of total peak(s) area corresponding tothe acidic species were quantified (FIG. 24). The percentage of acidicspecies in the control sample was as high as 26.6%. In the sample withthe highest tested concentration of lysine in this experiment (11 g/L)the percentage of acidic species was reduced to 18.1%. A dose dependentdecrease in acidic species was observed in test conditions with highertotal concentrations of lysine.

Additional experiments were performed with multiple cell lines inchemically defined or hydrolysate based media to demonstrate the widerange of applicability of this method. The experimental setup for eachof these experiments was similar to that described above and inmaterials and methods section. The summaries of results of the differentexperiments performed for adalimumab are summarized in FIGS. 25, 26, and27. A reduction in acidic species with increased lysine concentrationwas also observed in each case.

In addition to adalimumab, the utility of this method for acidic speciesreduction was also demonstrated for processes involving two other mAbs.The experimental setup for each of these experiments was similar to thatdescribed above and in the materials and methods section. The reductionof acidic species with lysine addition for experiments corresponding toeach mAb is summarized in FIGS. 28, 29. For mAb2, a significantreduction in acidic species was observed at lysine concentration of 11g/L.

In U.S. Application Ser. No. 61/893,088, (the contents of which areincorporated herein by reference), the utility of lysine supplementationto culture media for the modulation of the lysine variant distributionis described. To estimate the acidic species reduction that isindependent of this redistribution of lysine variants, Protein A eluatesamples from a representative set of lysine supplementation experimentswere pre-treated with the enzyme carboxypeptidase before WCX-10. One setof samples from an adalimumab experiment and another set of samples froma mAb2 experiment were used for this analysis. The carboxypeptidasetreatment of the samples resulted in the cleavage of the C-terminallysine residues as demonstrated by the conversion of Lys1/Lys2 to Lys 0in each of these samples. As a result of this conversion, the acidicspecies quantified in these samples corresponded to an aggregate sum ofacidic species that would be expected to also include those species thatmay have previously shifted corresponding to the lysine variant shiftand perhaps gone unaccounted for in the samples that were not treatedwith carboxypeptidase prior to WCX-10. A dose dependent reduction inacidic species was observed in the carboxypeptidase treated samples withincreasing concentration of lysine for the adalimumab samples from 26.8%in the non-supplemented sample to 21.1% in the 10 g/L lysinesupplemented sample, a reduction of 5.7% in total acidic species (FIG.30). Similar results were also observed for the mAb2 samples (FIG. 31).This suggests that the acidic species reduction described here is notcompletely attributed to a probable shift of the acidic speciescorresponding to the lysine redistribution.

Effect of Histidine Supplementation to Cell Culture Media

The addition of histidine was tested in several experimental systemscovering multiple cell lines, media and monoclonal antibodies. Thefollowing is a detailed description of two representative experimentswhere two different cell lines (cell line 2 and cell line 3) werecultured in a chemically defined media (media 1) for the production ofadalimumab.

Cell line 2 was cultured in media 1 with different total concentrationsof histidine (0 (control), 4, 6, 8, 10 g/L). The cultures were performedin shake flasks in batch format with only glucose feed as described inthe materials and methods. The cells grew to maximum VCD in the range of12-22×10⁶ cells/ml for the different conditions tested. A dose dependentdecrease in viable cell density profile was observed with the 10 g/Lhistidine condition having significant reduction in growth (FIG. 32). Acorresponding effect on viability was also observed (FIG. 33). On Days10, 11 and 12 of culture samples were collected for titer analysis andreported for the harvest day for each sample (FIG. 34). There was asmall dose dependent decrease in titers for conditions with histidinesupplementation. On Days 11-12, duplicate shake flasks were harvestedand then subsequently analyzed using WCX-10 post Protein A purificationand the percentages of total peak(s) area corresponding to the acidicspecies were quantified (FIG. 35). The percentage of acidic species inthe control sample was as high as 26.5%. In the sample with the highesttested concentration of histidine in this experiment (10 g/L), thepercentage of acidic species was reduced to 15.6%. A dose dependentdecrease in acidic species was observed in test conditions withincreased histidine concentrations.

Cell line 3 was cultured in media 1 with different total concentrationsof histidine (0 (control), 2, 4, 6, 8 g/L). The cultures were performedin shake flasks in batch format with only glucose feed as described inthe materials and methods. The cells grew to maximum viable celldensities (VCD) in the range of 6-10×10⁶ cells/ml for the differentconditions tested. A dose dependent decrease in viable cell densityprofile was observed in all conditions with histidine concentrationshigher than that in the control (FIG. 36). The viability profiles weremore comparable between conditions with this cell line (FIG. 37). On Day12 of culture, samples were collected for titer analysis (FIG. 38). Thetiters for all conditions were comparable. On Day 12 of culture,duplicate shake flasks for each of the conditions were harvested andthen subsequently analyzed using WCX-10 post Protein A purification andthe percentages of total peak(s) area corresponding to the acidicspecies were quantified (FIG. 39). The percentage of acidic species inthe control sample was 26.2%. In the sample with the highest testedconcentration of histidine in this experiment (8 g/L), the percentage ofacidic species was reduced to 20.0%. A dose dependent decrease in acidicspecies was observed in test conditions with increased histidineconcentration.

Additional experiments were performed with multiple cell lines inchemically defined or hydrolysate based media to evaluate the wide rangeof applicability of this method. The experimental setup for each ofthese experiments was similar to that described above and in thematerials and methods section. The summaries of results of the differentexperiments performed for adalimumab are set forth in FIGS. 40, 41, and42. A reduction in acidic species with increased histidine concentrationwas observed with cell line 1 in media 1 (FIG. 40) and with cell line 2in media 3 (FIG. 42). For cell line 2 in media 3, a dose dependentreduction in acidic species was observed up to 4 g/L histidine, with nofurther significant reduction at higher concentrations of histidine(FIG. 42). For cell line 1, media 2, no significant reduction of acidicspecies was observed within the histidine concentration range (0-4 g/L)(FIG. 41).

In addition to adalimumab, the utility of this method for acidic speciesreduction was also demonstrated for processes involving two other mAbs.The experimental setup for each of these experiments was similar to thatdescribed above and in the materials and methods section. The reductionof acidic species with increased histidine concentration for experimentscorresponding to each mAb is summarized in FIGS. 43 and 44. For mAb2, incontrast with the results reported with arginine and lysinesupplementation shown previously, a clear significant dose dependentreduction in total acidic species from 28.1% in the control to 21.5% in4 g/L histidine sample was observed.

In U.S. Application Ser. No. 61/893,088, (the contents of which arehereby incorporated herein by reference), the utility of increasedhistidine to culture media towards modulation of the lysine variantdistribution is described. To estimate the acidic species reduction thatis independent of this redistribution of lysine variants, Protein Aeluate samples from a representative set of histidine supplementationexperiments were also pre-treated with the enzyme carboxypeptidasebefore WCX-10. One set of samples from adalimumab experiment and anotherset of samples from a mAb2 experiment were used for this analysis. Thecarboxypeptidase treatment of the samples resulted in the cleavage ofthe C-terminal lysine residues as demonstrated by the completeconversion of Lys 1/Lys2 to Lys 0 in each of these samples (data notshown here). A dose dependent reduction in acidic species was observedin the carboxypeptidase treated samples with increasing concentration ofhistidine (FIGS. 45 and 46). This indicates that the acidic speciesreduction described here is not completely attributed to a probableshift of the acidic species corresponding to the lysine redistribution.

Effect of Ornithine Supplementation to Cell Culture Media

The addition of ornithine was tested in several experimental systemscovering multiple cell lines, media and monoclonal antibodies. Thefollowing is a detailed description of two representative experimentswhere two different cell lines (cell line 2 and cell line 3) wereemployed in a chemically defined media (media 1) for the production ofadalimumab.

Cell line 2 was cultured in media 1 with different total concentrationsof ornithine (0 (control), 4, 6, 8, 10 g/L). The cultures were performedin shake flasks in batch format with only glucose feed as described inthe materials and methods. The cells grew to maximum VCD in the range of15-22×10⁶ cells/ml for the different conditions tested. A slightdecrease in viable cell density with ornithine supplementation wasobserved (FIG. 47). Corresponding differences in the viability profileswere also observed (FIG. 48). On Day 11 of culture, samples werecollected for titer analysis (FIG. 49). The titers for all conditionswere comparable. On Day 11, duplicate shake flasks were harvested foreach condition and then subsequently analyzed using WCX-10 post ProteinA purification and the percentages of total peak(s) area correspondingto the acidic species were quantified (FIG. 50). The percentage ofacidic species in the control sample was 26.5%. In the sample with thehighest tested concentration of ornithine in this experiment (10 g/L),the percentage of acidic species was reduced to 16.1%. A dose dependentdecrease in acidic species was observed in test conditions withincreased ornithine concentration.

Cell line 3 was cultured in media 1 supplemented with different totalconcentrations of ornithine (0 (control), 2, 4, 6, 8 g/L). The cultureswere performed in shake flasks in batch format with only glucose feed asdescribed in the materials and methods. The cells grew to maximum viablecell densities (VCD) in the range of 9.5-11.5×10⁶ cells/ml for thedifferent conditions tested. The viable cell density and viabilityprofiles were comparable (FIGS. 51 and 52). On Day 12 of culture,samples were collected for titer analysis (FIG. 53). The titers for allconditions were comparable. On Day 12 of culture, duplicate shake flasksfor each of the conditions were harvested and then subsequently analyzedusing WCX-10 post Protein A purification and the percentages of totalpeak(s) area corresponding to the acidic species were quantified (FIG.54). The percentage of acidic species in the control sample was 24.8%.In the sample with the highest tested concentration of ornithine in thisexperiment (8 g/L), the percentage of acidic species was reduced to20.5%. A dose dependent decrease in acidic species was observed in testconditions with increased ornithine concentration.

Additional experiments were performed with multiple cell lines inchemically defined or hydrolysate based media to evaluate the wide rangeof applicability of this method. The experimental setup for each ofthese experiments was similar to that described above and in thematerials and methods section. The summaries of results of the differentexperiments performed for adalimumab are summarized in FIGS. 55, 56 and57. For cell line 1 in media 1, a dose dependent reduction was observed(FIG. 55). However, for cell line 1 in media 2, a hydrolysate media, nosignificant reduction in acidic species was observed across theconditions (FIG. 56). For cell line 2 in media 3, a reduction in acidicspecies from 22.1% in the control sample to 18.7% in the 2 g/L ornithinesample with no further reduction at higher ornithine concentrations wasobserved (FIG. 57).

In addition to adalimumab, the utility of this method for acidic speciesreduction was also demonstrated for processes involving two other mAbs.The experimental setup for each of these experiments was similar to thatdescribed in the section above and in the materials and method section.The reduction of acidic species with ornithine addition for experimentscorresponding to each mAb is summarized in FIGS. 58 and 59. In the caseof mAb1, a 7.3% dose dependent reduction in total acidic species wasobserved within the concentration range tested. For mAb2, about 2%reduction was observed in the 1 g/L ornithine concentration sample withminimum further reduction at higher ornithine concentrations.

Similar to the analysis conducted with the other amino acids, Protein Aeluate samples from a representative set of ornithine experiments werealso pre-treated with the enzyme carboxypeptidase before WCX-10. One setof samples from adalimumab experiment and another set of samples from amAb2 experiment were used for this analysis. A dose dependent reductionin acidic species was observed in the carboxypeptidase treated sampleswith increasing concentration of ornithine (FIGS. 60 and 61). Thepercentage of acidic species was also comparable between an untreatedand a carboxypeptidase treated sample for a particular concentration ofornithine. This indicates that the acidic species reduction isindependent of any probable shift of the acidic species that may becorresponding to any lysine redistribution.

Effect of Increasing a Combination of Arginine, Lysine, Histidine,Ornithine to Cell Culture Media

In this experiment, the combined use of the four amino acids arginine,lysine, histidine and ornithine for acidic species reduction isdemonstrated. The experiment described here was performed usingadalimumab producing cell line 2 in chemically defined media (media 1).The concentration range for arginine and lysine in this experiment was1-3 g/L while the concentration range for histidine and ornithine inthis experiment was between 0-2 g/L. In comparison to the lowerconcentrations, or conditions where a single amino acid concentrationwas increased, a further reduction in total acidic species was observedin conditions where combinations of amino acids were increased in themedia (FIG. 62). A progressive decrease was observed in total acidicspecies when more amino acids were increased in combination. Thepercentage of acidic species was reduced from 21.9% in the lowestconcentration sample to 12.3% in the sample with high concentrations ofall four amino acids.

Control of Acidic Species Through Cell Culture with Increased Arginineand Lysine and Choice of Harvest Criterion and/or Modulation of pH

The increase of the amino acid (arginine, lysine) concentration in basalmedia may also be combined with choice of when to harvest a culture toachieve optimal reduction in total acidic species. In this example, astudy was carried out in 3 L bioreactors with cell line 1 (producingadalimumab) in media 1. Two sets of conditions were tested: controlcondition (arginine 1 g/L, lysine 1 g/L); Test condition 1 (arginine 3g/L, lysine 5 g/L). Cell growth, viability and titer profiles werecomparable between the conditions (FIGS. 63, 64, and 65). A small amountof cell culture harvests were collected every day from day 4 to day 10from each of the reactors and submitted for Protein A purification andWCX-10 analysis. The percentage of acidic species in the controlcondition increased from 12.1% (on day 4) to 24.6% (on day 10) (FIG.66). The percentage of acidic species in the test condition 1 was lowerthan that observed in the control condition at each correspondingculture day. The percentage of acidic species in the test condition alsoincreased from 8.7% (day 4) to 18.8% (day 10). The rate of increase inacidic species with culture duration also correlated with the drop inviability for both conditions, with a sharp increase on day 8. Thus,along with increasing arginine and lysine concentrations in culturemedia, choice of harvest day/harvest viability can be used incombination to achieve a desired acidic species reduction.

The increase of the amino acid (arginine, lysine) concentration in basalmedia may be combined with process pH modulation to achieve furtherreduction in total acidic species. In this example, a study was carriedout in 3 L bioreactors with cell line 1 (producing adalimumab) inmedia 1. Three sets of conditions were tested in duplicates: Controlcondition (arginine (1 g/L), lysine (1 g/L), pH 7.1->6.9 in 3 days, pH6.9 thereafter); Test condition 1 (arginine (3 g/L), lysine (3 g/L), pH7.1->6.9 in 3 days, pH 6.9 thereafter); Test condition 2 (arginine (3g/L), lysine (3 g/L), pH 7.1->6.8 in 3 days, pH 6.8 thereafter). Incomparison to the control, a slight decrease in VCD profile and harvesttiter was observed for condition 2 (FIGS. 67, 68, and 69). The cultureswere harvested when the viability was less than 50% and the cultureharvests were submitted for Protein A and WCX-10 analysis. Thepercentage of acidic species in the control sample was 19.1%. Thepercentage of acidic species was reduced to 14.3% in test condition 1and to 12.8% in test condition 2 (FIG. 70). Thus, this demonstrates thatthe increase of amino acid concentration along with choice of lowerfinal process pH can be used in combination for further reducing theextent of acidic species.

Effect of Supplementation of CaCl₂ to Cell Culture Media

The addition of calcium chloride was tested in several experimentalsystems covering multiple cell lines, media and monoclonal antibodies.Following is a detailed description of two representative experimentswhere two different cell lines (cell line 2 and cell line 3) werecultured in a chemically defined media (media 1) for the production ofadalimumab.

Cell line 2 was cultured in media 1 with different concentrations ofcalcium (0.14, 0.84 and 1.54 mM). The cultures were performed in shakeflasks in batch format with only glucose feed as described in thematerials and methods. The cells grew to maximum viable cell densities(VCD) in the range of 22-24.5×10⁶ cells/ml for the different conditionstested. The viable cell density and viability profiles for all testconditions were comparable (FIGS. 71 and 72). On Day 10 of culturesamples were collected for titer analysis (FIG. 73). The titers for allconditions were comparable. On Day 10 duplicate shake flasks wereharvested for each condition and then subsequently analyzed using WCX-10post Protein A purification and the percentages of total peak(s) areacorresponding to the acidic species were quantified (FIG. 74). Thepercentage of acidic species in the 0.14 mM calcium condition was 23.8%.In the sample with the highest tested concentration of calcium in thisexperiment (1.54 mM), the percentage of acidic species was reduced to21.6%. A dose dependent decrease in acidic species was observed in testconditions with increased calcium concentration.

Cell line 3 was cultured in media 1 with different total concentrationsof calcium (0.14, 0.49, 0.84, 1.19, 1.54, 1.89 g/L). The cultures wereperformed in shake flasks in batch format with only glucose feed asdescribed in the materials and methods. The cells grew to maximum viablecell densities (VCD) in the range of 9.5-10.5×10⁶ cells/ml for thedifferent conditions tested. The viable cell density and viabilityprofiles for all test conditions were comparable (FIGS. 75 and 76). OnDay 11 of culture, samples were collected for titer analysis. Theharvest titers for all conditions were comparable (FIG. 77). On Day 11of culture, duplicate shake flasks for each of the conditions wereharvested and then subsequently analyzed using WCX-10 post Protein Apurification and the percentages of total peak(s) area corresponding tothe acidic species were quantified (FIG. 78). The percentage of acidicspecies in the 0.14 mM calcium condition was 23.7%. In the sample withthe highest tested concentration of calcium in this experiment (1.89mM), the percentage of acidic species was reduced to 20.7%. A dosedependent decrease in acidic species was observed in test conditionswith increased calcium concentration.

Additional experiments were performed with multiple cell lines inchemically defined or hydrolysate based media to evaluate the wide rangeof applicability of this method. The experimental setup for each ofthese experiments was similar to that described in the section above andin the materials and methods section. The summaries of results of thedifferent experiments performed for adalimumab are summarized in FIGS.79, 80 and 81. A reduction in acidic species with increased calciumconcentration was also observed in each case.

In addition to adalimumab, the utility of this method for acidic speciesreduction was also demonstrated for processes involving two other mAbs.The experimental setup for each of these experiments was similar to thatdescribed above. The dose dependent reduction of acidic species withornithine addition for experiments corresponding to each mAb issummarized in FIGS. 82 and 83. For mAb1, a small yet significant acidicspecies reduction from 15.4% (0.14 mM calcium sample) to 11.8% (1.54 mMcalcium chloride supplemented sample) was observed. For mAb2, a largerdose dependent reduction from 28.9% (0.14 mM calcium sample) to 23.1%(1.40 mM calcium chloride supplemented sample) was observed.

Effect of Increased Concentration of Arginine, Lysine, Calcium Chloride,Niacinamide in Combination

In this experiment, the effect of the combined use of the amino acidsarginine, lysine, inorganic salt calcium chloride and vitaminniacinamide for acidic species reduction was evaluated. The experimentdescribed here was performed using cell line 2 (producing adalimumab) inchemically defined media (media 1) supplemented with 3% (v/v) PFCHO(proprietary chemically defined medium formulation from SAFC). A centralcomposite DOE experimental design was used in this experiment. The basalmedia for each condition was supplemented with different concentrationsof the four supplements. Cell cultures were carried out in duplicatesfor each condition. Upon harvest, WCX-10 analysis was performed postProtein A purification. In Table 4, below, the experimental conditionsfrom DOE design, including the concentration of each componentsupplemented, and the % total acidic species (or AR) obtained for eachcondition is summarized. Reduction of acidic species through theincreased concentration of these components in combination was observed.For instance, condition (#24), where all four components were at theirmaximum concentration, the % total AR was reported to be reduced to9.7%. Using the data from the experiment, a model predicting the effectsof addition of these components to media for AR reduction (R²: 0.92,P<0.0001) is described in FIG. 84. The model predicted a contributionfrom each of the four components towards acidic species reduction. Itmay be also possible to utilize this model to predict the choice ofconcentrations of these different components to the media, in order toachieve a target reduction in total AR.

TABLE 4 Experimental design and summary for the combined addition ofarginine, lysine, calcium chloride and niacinamide Calcium ArginineLysine Chloride Niacinamide % Total Conditions (g/l) (g/l) (mM) (mM) AR1 0.0 4.0 0.7 0.8 13.0 2 0.0 6.0 1.4 0.0 12.6 3 4.0 2.0 0 1.6 12.3 4 4.06.0 0 1.6 11.6 5 2.0 4.0 0.7 0.8 11.2 6 0.0 6.0 0 0.0 15.0 7 0.0 6.0 1.41.6 10.7 8 0.0 2.0 0 0.0 16.7 9 2.0 4.0 0.7 0.8 11.0 10 4.0 6.0 1.4 1.611.0 11 2.0 2.0 0.7 0.8 12.9 12 2.0 4.0 1.4 0.8 11.1 13 0.0 6.0 0 1.613.2 14 4.0 2.0 0 0.0 12.3 15 2.0 4.0 0.7 0.0 13.0 16 2.0 4.0 0.7 1.611.4 17 0.0 2.0 1.4 1.6 12.0 18 2.0 4.0 0 0.8 12.0 19 4.0 4.0 0.7 0.812.0 20 0.0 2.0 1.4 0.0 14.0 21 4.0 6.0 1.4 0.0 11.0 22 0.0 2.0 0 1.613.6 23 2.0 6.0 0.7 0.8 11.0 24 4.0 2.0 1.4 1.6 9.7 25 4.0 6.0 0 0.011.8 26 4.0 2.0 1.4 0.0 10.4 27 2.0 4.0 0 0.0 12.7

Use of Niacinamide Supplementation to Cell Culture Media for AcidicSpecies Reduction

In addition to the use of niacinamide in combination with othersupplements described in the previous section, niacinamide addition mayalso be used independent of the other supplements as demonstrated in theexperiments below for two mAbs: adalimumab and mAb1.

For the experiment corresponding to adalimumab, cell line 1 was culturedin media 1 supplemented with different amounts of niacinamide (0, 0.2,0.4, 0.8 and 1.6 mM). The cultures were performed in shake flasks inbatch format with only glucose feed as described in the materials andmethods. The cells grew to maximum VCD in the range of 8.5-11×10⁶cells/ml for the different conditions tested. A slight decrease in theviable cell density profile was observed with the maximum niacinamidesupplementation (1.6 mM for this experiment) (FIG. 85). The viabilityprofile for the test conditions were comparable (FIG. 86). On Day 12 ofculture, samples were collected for titer analysis. The titers for allconditions were comparable (FIG. 87). On Day 11 and day 12, duplicateshake flasks were harvested for each condition and then subsequentlyanalyzed using WCX-10 post Protein A purification and the percentages oftotal peak(s) area corresponding to the acidic species were quantified(FIGS. 88 and 89). The percentage of acidic species in the day 10control sample (without niacinamide supplementation) was 19.6%. In theday 10 sample with the highest tested concentration of niacinamide inthis experiment (1.6 mM), the percentage of acidic species was reducedto 15.9%. Similar acidic species reduction with niacinamidesupplementation was also observed in the day 12 samples.

For the experiment corresponding to mAb2, a mAb2 producing cell line wascultured in media 1 supplemented with different amounts of niacinamide(0, 0.1, 0.5, 1.0, 3.0 and 6.0 mM). The cultures were performed in shakeflasks in batch format with only glucose feed as described in thematerials and methods. The cells grew to maximum viable cell densities(VCD) in the range of 14-21.5×10⁶ cells/ml for the different conditionstested. A slight decrease in the viable cell density profile wasobserved for the conditions with 3.0 mM and 6.0 mM niacinamideconcentrations (FIG. 90). The viability profiles for all test conditionswere comparable (FIG. 91). On Day 12 of culture samples were collectedfor titer analysis (FIG. 92). The titers for all conditions werecomparable. On Day 12 duplicate shake flasks were harvested for eachcondition and then subsequently analyzed using WCX-10 post Protein Apurification and the percentages of total peak(s) area corresponding tothe acidic species were quantified (FIG. 93). The percentage of acidicspecies in the control sample (without niacinamide supplementation) was27.0%. In the sample with the highest tested concentration ofniacinamide in this experiment (6.0 mM), the percentage of acidicspecies was reduced to 19.8%. A dose dependent decrease in acidicspecies was observed in test conditions with niacinamidesupplementation.

Supplementation of Basic Amino Acids Arginine and Lysine to Cell CultureMedia for Reduction of Methylglyoxal (MGO) Modification of Antibody

In this experiment, the effect of MGO modification on acidic speciesreduction was examined. Adalimumab producing cell line 1 was cultured ina chemically defined media (media 1) which was supplemented with aminoacids, as described below.

Materials and Methods

Cell Source and Adaptation Cultures

Cells were cultured in their respective growth media (chemically definedmedia (media 1)) in a combination of vented non-baffled shake flasks(Corning) on a shaker platform at 110 RPM (cell line 1), and 10 L or 20L wave bags (GE). Cultures were propagated in a 35° C., 5% CO₂ incubatorin order to obtain the required number of cells to initiate productionstage cultures.

Cell Culture Media

For preparation of media 1, the media (IVGN GIA-1, a proprietary basalmedia formulation from Invitrogen) was supplemented with L-glutamine,sodium bicarbonate, sodium chloride, and methotrexate solution.Production media consisted of all the components in the growth medium,excluding methotrexate. Both growth and production medium were alsosupplemented with insulin.

Amino acids used for the experiments (arginine (Sigma, A8094) and lysine(Calbiochem, 4400)) were reconstituted in Milli-Q water to make a 100g/L stock solution, which was subsequently supplemented to both growthand production basal media. After addition of amino acids, media wasbrought to a pH similar to unsupplemented (control) media using 6Nhydrochloric acid/5N NaOH, and it was brought to an osmolality similarto unsupplemented (control) media by adjusting the concentration ofsodium chloride.

All media was filtered through Corning 1 L filter systems (0.22 μm PES)and stored at 4° C. until usage.

Production cultures were initiated in 3 L Bioreactors (Applikon). Forthe bioreactor experiments, 3 L bioreactors (1.5 L working volume) wererun at 35° C., 30% DO, 200 rpm, pH set-point of 7.1. The cells weretransferred from the seed train to the production stage at a split ratioof 1:5.

Cultures were run in either batch mode and were cultured in therespective production medium (media 1 supplemented with arginine (4 g/L)or lysine (4 g/L)). 1.25% (v/v) of 40% glucose stock solution was fedwhen the media glucose concentration reduced to less than 3 g/L.

Retention samples for titer analysis, of 2×1.5 mL, were collected dailybeginning on Day 8, and frozen at −80° C. The samples taken from eachwere later submitted for titer analysis.

The harvest procedure of the shake flasks and reactors involvedcentrifugation of the culture sample at 3,000 RPM for 30 min and storageof supernatant in PETG bottles at −80° C. before submission for ProteinA purification and WCX-10 analysis.

WCX-10 Assay

The WCX-10 assay method was employed as described above in the Materialsand Methods section.

Lysine-C Peptide Mapping for MGO Quantification

The procedure for lysine-C peptide mapping for MGO quantification wascarried out as described above in the Materials and Methods section.

Results and Discussion

The majority of cultures grew to a similar peak VCD in the range of9-10×10⁶ cells/mL (FIG. 94A). The viability profiles of the cultureswere also comparable with harvest viabilities between 10-25%. Theculture duration (10 days) was similar between the conditions (FIG.94B).

Using WCX-10 analysis on harvest samples post Protein A purification,the percentages of total peak(s) area corresponding to the acidicspecies were quantified. The percentage of acidic species in the controlsample was 36.5%. In the samples from cultures supplemented witharginine and lysine, the percentage of total acidic species was reducedto 20.1% and 28.0%, respectively (FIG. 94C). Significant reduction in %AR1 was also observed in these cultures: from 16.8% in the controlsamples to 7.3% (arginine supplemented cultures) and 12.8% (lysinesupplemented cultures) (FIG. 94C). The extent of MGO modification wasalso quantified using the Lys-C peptide mapping and reported as thepercentage of MGO modified peptides among those that are moresusceptible to MGO modification. From these results, it is apparent that% MGO modification was also significantly reduced in the culturessupplemented with the amino acids (FIG. 94D).

Example 2 Method for Reducing the Extent of Acidic Species in CellCulture by Adjusting Process Parameters

The experiments described below in the instant Example demonstrate thataltering cell culture process parameters on-line can be used to modulateand/or reduce the acidic species of a protein of interest, e.g., theantibody adalimumab or mAb2. For example, an increased dissolved oxygenconcentration and/or a decrease in final pH can lead to reductions inAR.

Materials and Methods

Cell Source and Adaptation Cultures

Two adalimumab producing CHO cell lines (cell line 1 and cell line 3)and a mAb2 producing cell line were employed in the studies covered inthis Example. Upon thaw, adalimumab producing cell line 3 was culturedin chemically defined growth media (media 1) in a combination of ventedshake flasks on a shaker platform at 140 rpm and 20 L wave bags.Cultures were propagated in a 36° C., 5% CO₂ incubator to obtain therequired number of cells to be able to initiate production stagecultures.

Upon thaw, adalimumab producing cell line 1 was cultured in ahydrolysate based growth media (media 2) in a combination of ventedshake flasks on a shaker platform at 110 rpm and 20 L wavebags in a 35°C., 5% CO₂ incubator. In some cases, the culture might be transferredinto a seed reactor with pH 7.1, 35° C. and 30% DO. The culture would beadapted to either media 1 or media 2 by propagated in a 10 L or 20 Lwavebag for 7-13 days with one or two passages before initiatingproduction stage cultures.

Upon thaw, mAb2 producing cells were cultured in media 1 in acombination of vented non-baffled shake flasks (Corning) on a shakerplatform at 140 RPM and 20 L wave bags (GE). Cultures were propagated ina 35° C., 5% CO₂ incubator to obtain the required number of cells to beable to initiate production stage cultures.

Cell Culture Media

Media 1, the chemical defined growth or production media, was preparedfrom basal IVGN CD media (proprietary formulation). For preparation ofthe IVGN CD media formulation, the proprietary media was supplementedwith L-glutamine, sodium bicarbonate, sodium chloride, and methotrexatesolution. Production media consisted of all the components in the growthmedium, excluding methotrexate. For cell line 1 and mAb2, the medium wasalso supplemented with insulin. In addition, 10 mM or 5 mM of Galactose(Sigma, G5388) and 0.2 μM or 10 μM of Manganese (Sigma, M1787) weresupplemented into production medium for cell line 3 or 1, respectively.Osmolality was adjusted by the concentration of sodium chloride. Allmedia was filtered through filter systems (0.22 μm PES) and stored at 4°C. until usage.

Media 2 is the hydrolysate based media, which contains basal proprietarymedia, Bacto TC Yeastolate and Phytone Peptone.

Production Cultures

Production cultures were initiated in 3 L Bioreactors (Applikon). Thebioreactors (1.5-2.0 L working volume) were run at the followingconditions (except for the different experimental conditions): 35° C.,30% DO (dissolved oxygen), 200 rpm, pH profile from 7.1 to 6.9 in threedays and pH 6.9 thereafter. In all experiments, the cells weretransferred from the wavebag to the production stage at a split ratio of1:5.6 (except mAb2 with a ratio of 1:5). When the media glucoseconcentration reduced to less than 3 g/L, approximately 1.25% (v/v) of40% glucose stock solution was fed.

The harvest procedure of reactors involved centrifugation of the culturesample at 3,000 RPM for 30 min and storage of supernatant in PETGbottles at −80° C. before submission for Protein A purification andWCX-10 analysis.

WCX-10 Assay

The acidic species and other variants present in cell culture harvestsamples were quantified. Cation exchange chromatography was performed ona Dionex ProPac WCX-10, Analytical column (Dionex, Calif.). Foradalimumab producing cell lines, a Shimadzu LC10A HPLC system was usedas the HPLC. The mobile phases used were 10 mM Sodium Phosphate dibasicpH 7.5 (Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mMSodium Chloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B:0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B:28-34 min) was used with detection at 280 nm. The WCX-10 method used formAb B used different buffers. The mobile phases used were 20 mM(4-Morpholino) ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobilephase A) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B).An optimized gradient (minute/% B): 0/3, 1/3, 46/21, 47/100, 52/100,53/3, 58/3 was used with detection at 280 nm.

Quantitation is based on the relative area percent of detected peaks.The peaks that elute at relative residence time earlier than the mainpeak corresponding to the drug product are together represented as theacidic peaks.

Results

Effect of Process pH in Media 1 with Cell Line 1

Five different pH conditions were assessed in this study: 7.1, 7.0, 6.9,6.8 and 6.7. The cultures were started at pH set point of 7.1; then wereramped down to the target pH set points within 4 days. All culturesreached the same maximum viable cell density on day 8, except for theculture at pH 6.7 condition, in which the maximum cell density was muchlower than the other cultures (FIG. 95). In addition, the viability ofthe culture at pH 7.1 and pH 7.0 dropped much earlier than the othercultures. The viability of cultures at pH 7.1 and pH 7.0 were 38% and54% on day 10, respectively; while the viability of the cultures atlower pH (including pH 6.9, 6.8 and 6.7) was above 70% on the same day(FIG. 96). Samples taken on the last day of the cultures were measuredfor IgG concentration. The titer of each tested condition increasedcorresponding to the decrease in pH, from 1.2 g/L in the pH 7.1condition to 1.8 g/L in the pH 6.8 condition; however, product titer wasnot continued to increase at pH 6.7 (1.6 g/L) (FIG. 97). The cultureswere harvested either on day 10 or on day 12. The harvest was Protein Apurified, then analyzed using WCX-10. The resulting peak areas fromWCX-10 analysis were quantified (FIG. 98). The percentage of acidicspecies decreased corresponding to the decrease in pH, from 56.0% in thepH 7.1 condition to 14.0% in the pH 6.7 condition. Since the cultures atpH 6.9, 6.8 and 6.7 were at 70% viability on day 10, additional sampleswere taken on day 12 for these cultures, when viability reached ˜50%.WCX-10 analysis was also performed for these samples. The percentage ofacidic species on day 12 was increased for these three conditions (i.e.,pH 6.9, 6.8 and 6.7) comparing to day 10; however, the increase in thepercentage of acidic species was smaller at lower pH. The percentage ofacidic species increased 18.8% (pH 6.9), 8.1% (pH 6.8) and 3.5% (pH6.7), respectively from day 10 (70% viability) to day 12 (50%viability). Therefore, the percentage of acidic species was lower atlower pH on day 12 too. The percent acidic species decreased withdecrease in pH from 39.1% in the pH 6.9 condition to 17.5% in the pH6.7condition, for a total reduction of 21.6% on day 12.

The effect of process pH to specifically reduce particular acidicvariants within the larger fraction of total acidic species was alsoevaluated. In Table 5, a summary of the extent of some of thesub-species of the acidic species fraction have been presented. Alongwith the reduction in total acidic species, the methods presented inthis section may also be used for reduction of sub-species that include,but not limited to, AR1, AR2 and MGO modified product variants.

TABLE 5 Effect of process pH on reduction of sub-species of acidicvariants Sample % MGO modified species Final LIGHT CHAIN HEAVY CHAIN pH% AR % AR1 % AR2 Arg 30 Arg 93 Arg 108 Arg 16 (19) Arg 259 Arg 359 Arg420 TOTAL 7.1 56.0 32.8 23.3 26.1 10.6 0.2 6.1 2.7 3.5 0.5 49.7 6.9 39.118.9 20.2 9.5 3.8 0.0 2.2 0.9 1.2 0.2 18.8 6.7 17.5 5.2 12.2 1.2 0.5 0.00.2 0.1 0.1 0.0 2.0

Effect of Process pH in Media 2 with Cell Line 1

Three different pH conditions were assessed in this study: 7.0, 6.9, and6.8. The cultures were started at pH of 7.1; then were ramped down tothe target pH set points within 3 days of culture. The viable celldensity and viability were comparable across the different pH set pointsuntil day 8. After day 8, the viable cell density and viability wereslightly higher with lower pH set points (FIGS. 99 and 100). Thecultures were harvested on ˜50% viability. The product titer wasslightly higher at pH 6.8 comparing to pH 6.9 and 7.0 (FIG. 101). Theresulting peak areas from WCX-10 analysis were quantified (FIG. 102).The percentage of acidic species decreased with decrease in pH from20.7% in the pH 7.0 condition to 18.1% in the pH6.8 condition, for atotal reduction of 2.6%.

Effect of Process pH in Media 1 with Cell Line 3

Five different pH conditions were assessed in this study: 7.1 7.0, 6.9,6.8, and 6.7. The cultures were started at pH set point of 7.1; thenwere ramped down to the target pH set points within 4 days of culture.The pH set points showed significant effect on the cell growth andviability with this cell line and media. Cell density was lower athigher pH and viability also dropped earlier at higher pH (FIGS. 103 and104). The cells were harvested either on day 10 or when viabilitydropped to equal or less than 50%. The titer was slightly increased asthe pH was reduced, reached the highest titer at pH 6.8 condition (FIG.105). The resulting peak areas from WCX-10 analysis were quantified(FIG. 106). The percent acidic species decreased with decrease in pHfrom 29.7% in the pH 7.1 condition to 21.5% in the pH6.7 condition, fora total reduction of 8.2%.

Effect of Dissolved Oxygen (DO) in Media 2 with Cell Line 1 at 35° C.

Three different dissolved oxygen (DO) conditions were assessed in thisstudy: 20%, 30% and 50%. The cultures were set at 35° C. The celldensity and viability were very comparable at different DO conditions(FIGS. 107 and 108). The cultures were harvested at the target viabilityof 50% for each condition. The harvest titer was higher at 50% DOcompared to 20% DO (FIG. 109). The harvest was also taken throughProtein A purification before WCX-10 analysis. The percentage of acidicspecies in each of the test conditions was 20.6% (20% DO), 19.0% (30%DO), and 17.7% (50% DO), respectively (FIG. 110). The percentage ofacidic species was in general lower at higher dissolved oxygenconcentrations. The percentage of acidic species decreased with increasein DO from 20.6% in the 20% DO condition to 17.7% in the 50% DOcondition, for a total reduction of 2.9%.

Effect of Dissolved Oxygen (DO) in Media 2 with Cell Line 1 at 33° C.

Three different DO conditions were assessed in this study: 20%, 30% and60%. The cell density, viability and product titer were very comparableat different DO condition (FIGS. 111, 112 and 113). The percentage ofacidic species in each of the test conditions was 20.1% (20% DO), 17.8%(30% DO), and 17.7% (60% DO), respectively (FIG. 114). The percentage ofacidic species was in general lower at higher dissolved oxygenconcentrations. The percentage of acidic species decreased with increasein DO from 20.1% in the 20% DO condition to 17.7% in the 60% DOcondition, for a total reduction of 2.4%.

Effect of Dissolved Oxygen (DO) in Media 1 with Cell Line 1 at 35° C.

Three different DO conditions were assessed in this study: 20%, 30% and50%. The cultures were set at 35° C. The cell density and viability werevery comparable at different DO conditions (FIGS. 115 and 116). Thecultures were harvested at the target viability of 40% for eachcondition. The harvest titer was higher at 30% and 50% DO comparing to20% DO (FIG. 117). The harvest was also taken through Protein Apurification before WCX-10 analysis. The percentage of acidic species ineach of the test conditions was 23.9% (20% DO), 22.4% (30% DO), and20.3% (50% DO), respectively (FIG. 118). The percentage of acidicspecies was in general lower at higher dissolved oxygen concentrations.The percentage of acidic species decreased with increase in DO from23.9% in the 20% DO condition to 20.3% in the 50% DO condition, for atotal reduction of 3.6%.

Effect of Dissolved Oxygen (DO) in Media 1 with Cell Line 3

The study was performed at four different temperature levels (33° C.,34° C., 35° C. and 36° C.) with two different DO conditions (20% DO and50% DO). In general, the cell growth at different dissolved oxygenlevels was similar except at 35° C., in which the cell density was lowerat 50% DO (FIG. 119). The cultures were harvested either on day 10 or at˜50% viability (FIG. 120). The titer at ˜50% viability is comparable atdifferent DO conditions (FIG. 121). The percentage of acidic species wasin general lower at higher dissolved oxygen at each tested temperaturecondition (FIG. 122). On day 10, the percentage of acidic speciesdecreased with increase in DO at 36° C. from 25.2% in the 20% DOcondition to 22.7% in the 50% DO condition, which is 2.5% of decrease;the percentage of acidic species decreased with increase in DO at 35° C.from 23.2% in the 20% DO condition to 19.4% in the 50% DO condition, fora total reduction of 3.8%; the percentage of acidic species decreasedwith increase in DO at 34° C. from 18.2% in the 20% DO condition to17.1% in the 50% DO condition, for a total reduction of 1.1% and thepercentage of acidic species decreased with increase in DO at 33° C.from 14.3% in the 20% DO condition to 12.9% in the 50% DO condition, fora total reduction of 1.4%. On day 12, when the viability was at ˜50% forthe 34° C. test conditions, the percentage of acidic species decreasedwith increase in DO from 21.5% in the 20% DO condition to 20.6% in the50% DO condition, for a total reduction of 0.9%. Lastly, on day 14, whenthe viability was at ˜50% for the 33° C. test conditions, the percentageof acidic species decreased with increase in DO from 19.7% in the 20% DOcondition to 17.9% in the 50% DO condition, for a total reduction of1.8%. In summary, at all tested temperature conditions on differentharvest days, the percentage of acidic species was lower at higherdissolved oxygen concentrations.

Effect of Dissolved Oxygen (DO) in Media 1 with mAb2

Six different DO conditions were assessed: 10%, 20%, 30%, 50%, 60% and80%. The cultures were set at 35° C. In general, the cell density,viability and titer at different dissolved oxygen levels were comparable(FIGS. 123, 124 and 125). The percentage of acidic species in each ofthe test conditions was estimated to be 26.5% (10% DO), 27.3% (20% DO),27.3% (30% DO), 25.8% (50% DO), 24.4% (60% DO) and 24.5% (80% DO),respectively (FIG. 126). The percentage of acidic species was in generallower at higher dissolved oxygen. The percentage of acidic speciesdecreased with increase in DO from 27.3% in the 20% DO condition to24.5% in the 80% DO condition, for a total reduction of 2.8%.

Example 3 Method for Reducing Acidic Species by the Addition of AminoAcids to Clarified Cell Culture Harvest and by Modifying the pH of theClarified Harvest

The present Example describes processes for reducing and controllinglevels of acidic species in antibody preparations. Specifically, thisExample provides a method for reducing the acidic variant content inclarified harvest, as well as a method for reducing the formation rateof acidic species in clarified harvest. The method involves addingadditives like various amino acids to clarified harvest or adjusting thepH of the clarified harvest using acidic substances.

As shown below, antibody acidic species in clarified harvest can bereduced by adding additives such as arginine or histidine to clarifiedharvest at concentrations of more than 100 mM and 50 mM, respectively.AR reduction can also be achieved by pH adjustment of the clarifiedharvest to pH 6 or pH 5. In addition, the rate of acidic variantformation can be reduced through the use of arginine or histidine in aconcentration dependent manner, or by low pH treatment of the clarifiedharvest.

Materials and Methods

Clarified Harvest Material

Different batches of adalimumab clarified harvest material were employedin the following experiments described below. Clarified harvest isliquid material containing a composition of interest, e.g., a monoclonalantibody of interest that has been extracted from a fermentationbioreactor after undergoing centrifugation to remove large solidparticles and subsequent filtration to remove finer solid particles andimpurities from the material. Clarified harvest was used for low pHtreatment studies described herein. Clarified harvest was also used forthe experiments to study the effect of amino acid concentration on thepresence of acidic species in clarified harvest, and for acid type-pHtreatment studies described herein. Different batches of mAb-B and mAb-Cclarified harvest material were employed for experiments to study theeffect of amino acid and low pH treatment studies on the presence ofacidic species described herein.

Preparation of Materials

The clarified harvest material was first adjusted to pH 4 using 3Mcitric acid. The material at pH 4 was then agitated for 60 minutesbefore adjusting the pH to a target pH of 5, 6 or 7 with 3M sodiumhydroxide. The material was then agitated for a further 60 minutes. Thesamples were then subjected to centrifugation at 7300×g for 15 minutesin a Sorvall Evolution RC with an SLA-3000 centrifuge bowl. Thesupernatants obtained from the centrifuged material were then depthfiltered using B1HC depth filters (Millipore) followed by 0.22 μmsterile filters. The filtrates of different pH were then subjected toholding for different period of time for evaluating the formation rateof acidic variants. After the holding, the material was purified withProtein A affinity column and the eluate was sampled and analyzed usingthe WCX-10 method. The preparation scheme is shown below in FIG. 127.

The material to study the effect of arginine on acidic species wasprepared in two ways. For lower target arginine concentrations of 5 mM,10 mM, 30 mM and 100 mM, they were made by adding the appropriate amountof 0.5M arginine stock buffer at pH 7 (pH adjusted with acetic acid) toattain the target arginine concentrations needed. For higher targetarginine concentrations of 50 mM, 100 mM, 300 mM, 500 mM, 760 mM, 1M and2M, they were made by adding the appropriate amount of arginine (solid)to the samples to attain the target arginine concentrations, withsubsequent titration to a final pH of 7 using glacial acetic acid.Arginine was adjusted to a final concentration of 100 mM using the twomethods to determine if the method of preparation would result indifferent effects. For all the experiments, following the arginineaddition, treated clarified harvests were held at room temperature forthe indicated duration followed by purification with Protein A columnand analysis of acidic variants. This study provided two results; (1)data of samples from Day 0 gave the effects of arginine on reducingacidic species in clarified harvest, (2) data of samples with differentholding days gave effect of arginine on reducing the formation rate ofacidic species. The preparation scheme is shown in FIG. 128.

The material to study the effect of histidine was prepared with targetconcentrations of 5 mM, 10 mM, 30 mM 50 mM, 100 mM, 200 mM and 250 mM.The samples were prepared by adding the appropriate amount of histidine(solid) to the samples to attain the target histidine concentrations,with subsequent titration to a final pH of 7 using glacial acetic acid.The sample preparation scheme is shown in FIG. 129.

The material to study the effect of lysine was prepared with targetconcentrations of 5 mM, 10 mM, 30 mM 50 mM, 100 mM, 200 mM, 300 mM, 500mM and 1000 mM. The samples were prepared by adding the appropriateamount of lysine hydrochloride (solid) to the samples to attain thetarget lysine concentrations, with subsequent titration to a final pH of7 using hydrochloric acid. The sample preparation scheme is shown belowin FIG. 130.

The material to study the effect of methionine was prepared with targetconcentrations of 5 mM, 10 mM, 30 mM 50 mM, 100 mM, 200 mM and 300 mM.The samples were prepared by adding the appropriate amount of methionine(solid) to the samples to attain the target methionine concentrations,with subsequent titration to a final pH of 7 using glacial acetic acid.The sample preparation scheme is shown in FIG. 131.

The material to study the effect of different amino acids was preparedwith different target concentrations for each of the 20 amino acidsevaluated as well as two controls using sodium acetate in place of anamino acid, and the other simply bringing the pH of the clarifiedharvest down to pH 7 using glacial acetic acid. The targetconcentrations for the amino acids are shown below in Table 6.

TABLE 6 Amino Acid Target Concentrations Concentration Amino Acid (mM)Alanine 100 Arginine 100 Asparagine 100 Aspartic Acid 30 Cysteine 100Glutamic 30 Acid Glutamine 100 Glycine 100 Histidine 100 Isoleucine 100Leucine 100 Lysine 100 Methionine 100 Phenylalanine 100 Proline 100Serine 100 Threonine 100 Tryptophan 30 Tyrosine 2 Valine 100 NaAc 100

The samples were prepared by adding the appropriate amount of amino acid(solid) to the samples to attain the target amino acid concentrations asshown in Table 6, with subsequent titration to a final pH of 7 usingglacial acetic acid. The sample preparation scheme is shown in FIG. 132.

The material to study the effect of additives other than amino acids wasprepared with different target concentrations for each of the additivesevaluated as well as a control in which sodium hydroxide was used inplace of arginine to bring the pH of the material to pH 10 beforeneutralizing it back to pH 7 with glacial acetic acid. The targetconcentrations for the additives are shown below in Table 7.

TABLE 7 Alternative Additive Target Concentrations Additive Low ConcHigh Conc Sucrose 0.1M 1M Trehalose 0.1M 1M Mannitol 4% w/v 10% w/vGlycerol 1% v/v 10% v/v PEG 1% w/v 2% w/v Tween80 0.5% v/v 2% v/v

The samples were prepared by adding the appropriate amount of additiveto the samples to attain the target amino acid concentrations as shownin Tables 6 or 7, with subsequent titration to a final pH of 7 usingglacial acetic acid.

The material to study the effect of the aforementioned methods on CDMclarified harvest was prepared using the following scheme shown in FIG.133.

The mAb B hydrolysate clarified harvest was used to study the effect ofthe aforementioned methods.

The mAb C hydrolysate clarified harvest was used to study the effect ofthe aforementioned methods.

Hold Studies for Treated Clarified Harvest

After the aforementioned sample preparations, the samples were placed inseparate sterile stainless steel containers for the purpose of holdingat either 4° C. or at room temperature. For each material, differentcontainers were used for each day of holding evaluated. For theacidified samples, the acidic variant compositions of the samples wereevaluated on days 0, 3, 7 and 14 of holding at either temperature. Forthe arginine containing materials, the acidic variant compositions ofthe samples were evaluated on days 0, 5 and 8 of holding at roomtemperature. For the histidine containing materials, the acidic variantcompositions of the samples were evaluated on days 0, 3 and 7 of holdingat room temperature.

Acid Type and pH Effects on Clarified Harvest

The effects of acid type, clarified harvest pH and arginine content onacidic variant reduction were evaluated in this study. The samples wereprepared in triplicates on 3 consecutive days to target arginineconcentrations of either 0 mM (no arginine added) or 500 mM, thentitrated with either glacial acetic acid, phosphoric acid, 3M citricacid or 6M hydrochloric acid to target pH values of either 5, 6 or 7.One other sample was prepared by adding a 2M arginine acetate pH 7 stockbuffer to clarified harvest to attain a target arginine concentration of500 mM. The sample preparation scheme is shown in FIG. 134.

Protein A Purification

Protein A purification of the samples was performed using a 5 mLrProtein A FF Hitrap column (GE Healthcare) at 10 g adalimumab/L resinloading and a operating flow rate of 3.4 mL/min. Five column volumes(CVs) of equilibration (1×PBS pH 7.4) is followed by loading of thesample, then washing of the column with equilibration buffer to removenon-specifically bound impurities, followed by elution of the proteinwith 0.1M Acetic acid, 0.15M sodium chloride.

The eluate samples were collected and neutralized to pH 6.9-7.2 with 1MTris pH 9.5 at 45-75 minutes after collection. The samples were thenfrozen at −80° C. for at least one day before thawing and subjecting toWCX-10 analysis.

Effects of Purification Method, Acid Concentration and Neutralization onClarified Harvest

The effects of purification methods with different types ofchromatography resins, acid concentration and pH neutralization onacidic variant reduction were evaluated in this study. The followingsamples were prepared as shown below in Table 8.

TABLE 8 Acid Concentration Sample Treatments Sample Treatment ControlNone 3M Citric Acid pH 6 Titrate to pH 6 with 3M Citric Acid 1M CitricAcid pH 6 Titrate to pH 6 with 1M Citric Acid Glacial Acetic Acid pH 6Titrate to pH 6 with Glacial Acetic Acid 3M Acetic Acid pH 6 Titrate topH 6 with 3M Acetic Acid 3M Citric Acid pH 5 Titrate to pH 5 with 3MCitric Acid 3M Acetic Acid pH 5 Titrate to pH 5 with 3M Acetic Acid 3MCitric Acid pH 5 to 7 Titrate to pH 5 with 3M Citric Acid, then 3M Tristo pH 7 3M Acetic Acid pH 5 to 7 Titrate to pH 5 with 3M Acetic Acid,then 3M Tris to pH 7

Each of the material made was then subjected to either Mabselect Sure orFractogel S capture in duplicate. The eluate samples are collected andneutralized to pH 6.9-7.2 with 1M Tris pH 9.5 at 45-75 minutes aftercollection. The samples are then frozen at −80° C. for at least one daybefore thawing and subjecting to WCX-10 analysis.

Acidic Variant Analysis (WCX-10 Assay)

Cation exchange chromatography was performed on a 4 mm×250 mm DionexProPac WCX-10 Analytical column (Dionex, Calif.). A Shimadzu LC10A HPLCsystem was used to perform the HPLC assay. The mobile phases used were10 mM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). Abinary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A,100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at280 nm.

Quantitation is based on the relative area percent of detected peaks.The peaks that elute at relative residence time less than that of thedominant Lysine 0 peak are together represented as the acidic variantpeaks (AR).

Results

Effect of Low pH Treatment with Subsequent Neutralization

The results of the low pH treatment with subsequent neutralization areshown below in FIGS. 135 and 136. FIG. 136 shows that the low pHtreatment with subsequent neutralization to pH 5 or 6 reduces the rateof acidic variant formation over time. However, there is no significantreduction in initial acidic variant content, as shown in FIG. 135.

Effect of Arginine Treatment

The results of the arginine treatment are shown in FIG. 137 and FIG.138. FIGS. 137 and 138 show that the sample preparation method resultedin different levels of acidic species in clarified harvest. Adding a0.5M arginine pH 7 stock buffer tends to increase acidic species, whileadding pure arginine with subsequent acetic acid titration to pH 7reduced acidic variants at arginine concentrations of greater than 100mM. Moreover, the effect due to treatment method is demonstrated whencomparing the two 100 mM arginine samples, which show an absolutedifference of 1% in acidic variants between the two methods.

FIG. 139 shows that the rate of acidic variant formation decreases withincreasing arginine concentration in clarified harvest, plateauing ataround concentrations of 500 mM arginine and higher. However, the twomethods of sample preparation do not result in significantly differentformation rate of acidic variants.

Effect of Histidine Treatment

The results of the histidine treatment are shown in FIG. 140 and FIG.141. Similar to arginine treatment effect, as shown in FIG. 149, whenhistidine was added to clarified harvest with subsequent pHneutralization with acetic acid, acidic variants were reduced athistidine concentrations higher than 50 mM. FIG. 141 shows that the rateof acidic variants formation decreases with increasing histidineconcentration in clarified harvest, plateauing at around concentrationsof 200 mM histidine and higher.

Effect of Lysine Treatment

The results of the lysine treatment are summarized in FIG. 142 and FIG.143. Similar to arginine treatment effect, as shown in FIG. 149, whenlysine was added to clarified harvest with subsequent pH neutralizationwith acetic acid, acidic variants were significantly reduced by ˜1% ormore. FIG. 153 shows that the rate of acidic variants formationdecreases with increasing lysine concentration in clarified harvest.

Effect of Methionine Treatment

The results of the methionine treatment are summarized below in FIGS.154 and 165. Similar to arginine treatment effect, as shown in FIG. 149,when methionine was added to clarified harvest with subsequent pHneutralization with acetic acid, acidic variants were significantlyreduced by ˜1% or more at concentrations of >10 mM. FIG. 145 shows thatthe rate of acidic variants formation is not affected significantly bymethionine presence in clarified harvest.

Effect of Other Amino Acid Treatment

The results of the treatments with the various amino acids aresummarized below in FIGS. 146 and 147. As shown in FIG. 146, theaddition of 14 amino acids including arginine, histidine, lysine andmethionine resulted in lower amounts of acidic variant content inclarified harvest. The addition of sodium acetate or the use of aceticacid also caused a reduction in acidic variant content as well. FIG. 147shows that the rate of acidic variants formation is reduced by severalamino acids including arginine, histidine, lysine, aspartic acid,glutamic acid, and leucine.

Effect of Alternative Additive Treatment

The results of the treatments with the other additives are summarizedbelow in FIGS. 148 and 149. As shown in FIG. 148, the addition of any ofthe additives did not result in lower acidic variant content inadalimumab hydrolysate clarified harvest. However, FIG. 149 shows thatthe rate of acidic variants formation is reduced by most of theadditives.

Effect of Low pH/Arginine Treatment on Adalimumab CDM Clarified Harvest

The results of CDM clarified harvest study are summarized below in FIGS.150 and 151. As shown in FIG. 150, low pH/arginine treatment did notresult in lower acidic variant content in adalimumab CDM clarifiedharvest. However, FIG. 151 shows that the rate of acidic variantsformation is reduced significantly by all the treatments.

Effect of Low pH/Arginine Treatment on mAb B Hydrolysate ClarifiedHarvest

The results of mAb B hydrolysate clarified harvest study are summarizedbelow in FIGS. 152 and 153. As shown in FIGS. 152 and 153, lowpH/arginine treatment results in both lower acidic variant content andslower rates of acidic variants formation in mAb B hydrolysate clarifiedharvest.

Effect of Low pH/Arginine Treatment on mAb C Hydrolysate ClarifiedHarvest

The results of mAb C hydrolysate clarified harvest study are summarizedbelow in FIGS. 154 and 155. As shown in FIGS. 154 and 155, lowpH/arginine treatment results in both lower acidic variant content andslower rates of acidic variants formation in mAb C hydrolysate clarifiedharvest.

Effect of Acid Type and pH

The results obtained from the acid type-pH study are summarized in FIG.156. Greater acidic species reduction is obtained at lower pH. Arginineaddition also reduces acidic species content further, but not to asignificant extent when taking the high concentrations (500 mM) usedinto consideration. The results also show that acidic species reductionof ˜1% can be achieved with the usage of an arginine acetate stockbuffer, although using pure arginine powder with subsequent acidtitration performs slightly better. With regard to acid type used for pHadjustment, there were no significant differences between differentacids observed.

Effect of Purification Method, Acid Concentration and Neutralization

The results obtained from the study are summarized in FIGS. 157, 158,159, and 160. FIGS. 157 and 158 indicate that when the acid used is ofhigher concentration, there is an decrease in acidic variant content inhydrolysate clarified harvest as compared to a lower concentration acidbeing used. FIGS. 159 and 160 show that when the clarified harvest issubjected to base neutralization to pH 7 after being treated with lowpH, there is an increase in acidic variant content. The figures alsoshow that the Fractogel resin is better able to clear acidic variantsthan Mabselect Sure.

Example 4 Method for Reducing AR in Cell Culture Using a ContinuousMedia Perfusion Technology

As demonstrated in Example 3, above, generation or formation of acidicspecies in a population of proteins may occur during the hold of theantibody in clarified harvest or spent media. Thus, the possibility ofenhanced stability of the product antibody or a reduction in acidicspecies generation was explored using a continuous/perfusion based cellculture technology. Control or reduction in the amount of acidic speciespresent in the population of proteins obtained at the end of cellculture can be accomplished by modifying the exchange rate of freshmedium into the bioreactor (or removal of spent medium with productantibody out of the bioreactor).

Materials and Methods

Cell Source

One adalimumab producing CHO cell line (cell line 1) was employed in thestudy covered here. Upon thaw, the vial was cultured in a chemicallydefined growth media (media 1) in a series of vented shake flasks on ashaker platform at 110 rpm in a 35° C., 5% CO₂ incubator. Cultures werepropagated to obtain a sufficient number of cells for inoculation of theperfusion cultibag.

Cell Culture Media

A chemically defined growth or production media was used in this study.For preparation of the media formulation, the proprietary media(Invitrogen) was supplemented with L-glutamine, sodium bicarbonate,sodium chloride, recombinant human insulin and methotrexate solution.Perfusion stage media consisted of all the components in the growthmedium, with the exception of a higher concentration of recombinanthuman insulin and the exclusion of methotrexate solution.

Perfusion Culture

The perfusion culture was carried out with the Sartorius BIOSTAT RM 20optical perfusion system (SN#00582112) in a Sartorius Cultibag RM 10 Lperfusion pro 1.2my (lot 1205-014) perfusion bag. The perfusion bag wasrun with a working culture volume of 1.5 L and operation conditions of;pH: 7.00, dissolved oxygen 30%, 25 rpm, 35° C., an air overlay of 0.3slpm and a CO₂ overlay of 15 sccm. pH control was initiated on day threeof the culture. pH was controlled with 0.5M sodium hydroxide and CO₂additions.

Perfusion was carried out by ‘harvesting’ spent culture through anintegrated 1.2 μm filter integrated into the perfusion cultibag. Freshmedia was added to the culture through a feed line at the same rate asthe harvest. Perfusion began on day four of the process at a rate of 1.0exchanges per day (ex/day). The perfusion rate was adjusted throughoutthe run to accommodate glucose needs, lactate accumulation and samplingplans. Perfusion cell-free harvest samples were collected at perfusionrates of 1.5, 3.0 and 6.0 exchange volumes/day on day 5-6 of perfusion.A fresh harvest bag was used for each harvest sample. The samples werethen purified using Protein A and analyzed using WCX-10 assay.

The perfusion culture was ended on day 8 of the process.

WCX-10 Assay

The acidic species and other charge variants present in cell cultureharvest samples were quantified. Cation exchange chromatography wasperformed on a Dionex ProPac WCX-10, Analytical column (Dionex, Calif.).

The mobile phases used were 10 mM Sodium Phosphate dibasic pH 7.5(Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mM SodiumChloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B: 0-20min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B:28-34 min) was used with detection at 280 nm. The WCX-10 method used formAb2 samples used different buffers. The mobile phases used were 20 mM(4-Morpholino) ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobilephase A) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B).An optimized gradient (minute/% B): 0/3, 1/3, 46/21, 47/100, 52/100,53/3, 58/3 was used with detection at 280 nm. Quantitation is based onthe relative area percent of detected peaks, as described above.

Results

Effect of Use of Perfusion Technology and Choice of Medium ExchangeRates on Acidic Species

Adalimumab producing cell line 1 was cultured in media 1 and thecultures were carried out as described in the materials and methods. Asdescribed in Table 8, the exchange rates were modified over a period of24 hrs between day 5 and day 6 to explore the influence of mediumexchange rates on the extent of acidic species. At a continuous mediumexchange rate of 1.5 volumes/day, the product antibody in spent mediumwas collected in a harvest bag over a period of 17 hrs. The harvest bagwas then exchanged with a new bag and the old bag was transferred to 4°C. Subsequently and in succession, the medium exchange rates wereincreased to 3 and 6 volumes/day and the product harvest was collectedover a time period of 5 and 2 hrs, respectively. After an overnight holdat 4° C., the three harvest samples were processed through Protein A andanalyzed for acidic species using WCX-10. The percentage of acidicspecies in the sample with a medium exchange rate of 1.5 volumes/day was8.1%. In the sample with the highest tested exchange rate in thisexperiment (6 volumes/day), the percentage of acidic species was reducedto 6%. An exchange rate dependent reduction in acidic species wasobserved in the three samples (Table 9). Reductions in differentsub-species within the acidic variants (AR1 and AR2) were also noted. Anincrease in volumetric productivity, with exchange rate, was alsoobserved.

TABLE 9 Effect of medium exchange rates in a perfusion bioreactor onacidic species Harvest bag Exchange rate Exchange time Volumetric StartTime (no. of working (for collection in Productivity (day, hrs:min)volumes/day) harvest bag) (hrs) (mg/l-hr) % Total AR % AR1 % AR2 Day 5,16:00 1.5 17 10.94 8.1 2.0 6.1 Day 6, 10:25 3 5 39.80 6.9 1.7 5.2 Day 6,15:25 6 2 69.50 6.0 1.3 4.7

Example 5 Method for Acidic Species Reduction Through the Use ofContinuous Perfusion Technology and Addition of Amino Acids to CultureMedium

As set forth above in Example 4, reduction in the amount of acidicspecies present in the population of proteins obtained at the end ofcell culture can be accomplished by modifying the exchange rate of freshmedium into the bioreactor (or removal of spent medium with productantibody out of the bioreactor). In this Example, the ability to furtherreduce acidic species through the use of high medium exchange rates incombination with supplementation of basic amino acids (arginine andlysine) to the culture medium is described.

Materials and Methods

Cell Source

An adalimumab producing CHO cell line (cell line 1) was employed. Uponthaw, the vial was cultured in a chemically defined growth media(media 1) in a series of vented shake flasks on a shaker platform at 110rpm in a 35° C., 5% CO₂ incubator. Cultures were propagated to obtain asufficient number of cells for inoculation of the perfusion cultibag.

Cell Culture Media

A chemically defined growth or production media was used in this study.For preparation of the media formulation, the proprietary media(Invitrogen) was supplemented with L-glutamine, sodium bicarbonate,sodium chloride, recombinant human insulin and methotrexate solution.Perfusion stage media consisted of all the components in the growthmedium, with the exception of a higher concentration of recombinanthuman insulin and the exclusion of methotrexate solution. Arginine andlysine were added as powders directly to the media solution. After theamino acid addition the pH was adjusted to that of the unsupplementedmedia using 5N NaOH and 5N HCL as necessary, and the osmolality wasadjusted to that of the unsupplemented media by varying theconcentration of sodium chloride.

Perfusion Culture

The perfusion culture was carried out with the Sartorius BIOSTAT RM 20optical perfusion system (SN#00582112) in a Sartorius Cultibag RM 10 Lperfusion pro 1.2my (lot 1205-014) perfusion bag. The perfusion bag wasrun with a working culture volume of 1.5 L and operation conditions of;pH: 7.00, dissolved oxygen 30%, 25 rpm, 35° C., an air overlay of 0.3slpm and a CO₂ overlay of 15 sccm. pH control was initiated on day threeof the culture. pH was controlled with 0.5M sodium hydroxide and CO₂additions.

Perfusion was carried out by ‘harvesting’ spent culture through anintegrated 1.2 μm filter integrated into the perfusion cultibag. Freshmedia was added to the culture through a feed line at the same rate asthe harvest. The perfusion rate was adjusted throughout the run toaccommodate glucose needs, lactate accumulation and sampling plans.Perfusion cell-free harvest samples were collected at perfusion rates of1.5, 3.0, 4.0, 6.0 and 8.0 exchange volumes/day on day 6-8 of perfusion.A fresh harvest bag was used for each harvest sample. The samples werethen purified using Protein A and analyzed using WCX-10 assay.

WCX-10 Assay

The acidic species and other charge variants present in cell cultureharvest samples were quantified. Cation exchange chromatography wasperformed on a Dionex ProPac WCX-10, Analytical column (Dionex, Calif.).

The mobile phases used were 10 mM Sodium Phosphate dibasic pH 7.5(Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mM SodiumChloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B: 0-20min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B:28-34 min) was used with detection at 280 nm. The WCX-10 method used formAb2 samples used different buffers. The mobile phases used were 20 mM(4-Morpholino) ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobilephase A) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B).An optimized gradient (minute/% B): 0/3, 1/3, 46/21, 47/100, 52/100,53/3, 58/3 was used with detection at 280 nm. Quantitation is based onthe relative area percent of detected peaks, as described above.

Results: Effect of Use of Perfusion Technology and Choice of MediumExchange Rates on Acidic Species

Adalimumab producing cell line 1 was cultured in media 1 and thecultures were carried out as described in the materials and methods. Theexchange rates were modified over a period of 2 days between day 6 andday 8 to explore the influence of medium exchange rates on the extent ofacidic species. At a continuous medium exchange rate of 1.5 volumes/day,the product antibody in spent medium was collected in a harvest bag overa period of 22 hrs. The harvest bag was then exchanged with a new bagand the old bag was transferred to 4° C. Subsequently and in succession,the medium exchange rates were increased to 3 and 6 volumes/day on day 7and to 4 and 8 volumes/day on day 8 and the product harvests werecollected and transferred to 4° C. The harvest samples were processedthrough Protein A and analyzed for acidic species using WCX-10. Thepercentage of acidic species in the control sample with a mediumexchange rate of 1.5 volumes/day was 7.7%. The percentage of acidicspecies in the arginine and lysine supplemented cell culture with amedium exchange rate of 1.5 volumes/day was 4.3%. In the sample with thehighest tested exchange rate in this experiment (8 volumes/day), thepercentage of acidic species was reduced to 5.4% in the control sample,and reduced to 3.0% in the arginine and lysine supplemented cell culturesample. An exchange rate dependent reduction in acidic species wasobserved in both the cultures (FIG. 161). Thus, the combination ofarginine/lysine supplementation to culture media along with exchangerate modulation can be used to further reduce AR.

Example 6 Upstream and Downstream Process Combinations to Achieve Target% AR or AR Reductions

Upstream and downstream process technologies, e.g., cell culture andchromatographic separations, of the inventions disclosed herein can becombined together or combined with methods in the art to provide a finaltarget AR value or achieve a % AR reduction. Upstream methods for ARreduction include, but are not limited to, those described in theinstant application. Downstream methods for AR reduction are alsodescribed herein. Exemplary upstream and downstream process technologiesinclude, but are not limited to: cell culture additives and conditions;clarified harvest additives and pH/salt conditions; mixed mode mediaseparations; anion exchange media separations; and cation exchange mediaseparations.

The instant Example demonstrates the combined effect of one or more ofthese technologies in achieving a target AR value or AR reduction,thereby facilitating the preparation of an antibody material having aspecific charge heterogeneity. Additional examples of combinations ofdownstream technologies and upstream technologies are provided herein.

In this Example, the combination of upstream and downstream methodsinvolves the reduction of acidic species in 3 L bioreactor cell culturessupplemented with arginine (2 g/L) and lysine (4 g/L) as has beenpreviously demonstrated in the instant application. The results of thatstrategy are summarized in Table 10. The total acidic species wasreduced from 20.5% in the control sample to 10.2% in sample fromcultures that were supplemented with the additives. In this study,adalimumab producing cell line 1 was cultured in media 1 (chemicallydefined media) supplemented with amino acid arginine (2 g/L) and lysine(4 g/L) in a 300 L bioreactor. On Day 12 of culture, the culture washarvested and then subsequently analyzed using WCX-10 post Protein Apurification and the percentages of total peak(s) area corresponding tothe acidic species were quantified. The percentage of acidic species wasestimated to be 9.1% in the 300 L harvest sample.

TABLE 10 AR levels achieved with use of upstream technologies 3 LBioreactor 300 L Bioreactor Arginine (2 g/L) + Arginine (2 g/L) +Control Lysine (4 g/L) Lysine (4 g/L) AR1 AR2 Total AR1 AR2 Total AR1AR2 Total (%) (%) AR (%) (%) (%) AR (%) (%) (%) AR (%) 6.3 14.2 20.5 2.67.6 10.2 2.4 6.7 9.1

The material produced by the 300 L Bioreactor employing arginine andlysine additions, that effectively reduced the AR levels to 9.1% waspurified using a downstream process employing Mixed Mode chromatographyas the primary AR reduction method.

Adalimumab was purified by a Protein A chromatography step followed witha low pH viral inactivation step. The filtered viral inactivatedmaterial was buffer exchanged and loaded onto a Capto Adhere column. TheFlow Through of Capto Adhere material was then purified with a HICcolumn with bind/elute mode as well as Flow Through mode. As shown inTable 11, AR reduction was achieved primarily with MM step, with somecontribution from other steps. The table also shows that additionalproduct related substances such as aggregates and process relatedimpurities such as HCP can be effectively reduced employing thesecombined technologies.

TABLE 11 Complete Downstream Process Train with Protein A Capture - AR,HMW and HCP reduction Process Yield (%) % AR reduction % HMW reductionHCP LRF Clarified Harvest 97.0% n/a n/a n/a Prt-A Eluate Pool 89.6% 0.061.87 Viral Inactivated Filtrate 99.7% No reduction 0.07 0.39 MM FT pool91.9% 2.26 0.83 1.63 HIC (B/E) Eluate 90.1% 0.40 0.22 1.41 NanofiltrateFiltrate 90.7% No reduction No reduction 0.15 BDS (B/E) 102.0% Noreduction No reduction 0.22 HIC FT-pool 98.5% 0.16 0.23 0.46 VF(FT)Filtrate 96.1% No reduction No reduction 0.10 BDS (FT) 103.8% Noreduction No reduction No reduction

As is evident from the above example, the MM method further reduced theAR levels, by 2.26%. Therefore upstream technologies for reduction canbe combined with downstream technologies to achieve AR levels/ARreduction.

Example 7 Anion Exchange (AEX) Chromatography Examples

Materials & Methods

Chromatography Method

Except where noted, the Materials and Methods described in connectionwith the instant example were also employed in Examples 8 and 9, below.

Pre-packed resin columns were used in the following experiments, exceptwhere specified. The column was equilibrated in a buffer system withappropriate pH and conductivity. The column load was prepared fromProtein A affinity chromatography eluates or concentrated CEXchromatography elutes by buffer exchange (if the eluates were withdifferent buffer components from the mixed mode target buffer system) oraddition of the stock solutions and/or water to obtain the target pH andconductivity as specified (if the eluates were with the same buffercomponents as the mixed mode target buffer system). The prepared loadmaterial was filtered and loaded on the column according to the targetload amount (g protein/L resin) as specified followed by washing withthe equilibration buffer or buffer similar to equilibration buffer withvolumes as specified. The column Flow Through/Wash were collected asfractions or as a pool. Mixed mode column was regenerated with 0.1 Macetic acid, 0.15 M NaCl pH3, or 0.1 M Acetic acid solution, pH 3, or asspecified. 1M NaOH solution was used for column cleaning.

Buffer Preparation Method

Buffers for AEX were prepared targeting specific ion concentration forthe anion by fixing the anion concentration (acid) to the target value,and adjusting the solution with the cationic component (base) to achievethe appropriate pH. For example, to prepare a 10 mM Acetate-Tris buffersolution, pH 8.7, glacial acetic acid was dissolved in water to a targetconcentration of 10 mM and adjusted with concentrated Tris-base to pH8.7. Also for example, to prepare a 10 mM Formate-Tris buffer solution,pH 8.7, formic acid was dissolved in water to a target concentration of10 mM and adjusted with concentrated Tris-base to pH 8.7.

Buffers for CEX were prepared targeting specific ion concentration forthe cation by fixing the cation concentration (base) to the targetvalue, and adjusting the solution with the anionic component (base) toachieve the appropriate pH. For example, to prepare a 140 mMTris-Formate buffer solution, pH 7.5, Tris base was dissolved in waterto a target concentration of 140 mM and adjusted with formic acid to pH7.5.

AR Reduction and Recovery Calculations

In general, the Flow Through/wash fractions were collected and analyzedwith WCX-10 method for AR levels. By actual or calculated pooling of thefractions the recovery and the corresponding AR levels were calculated.

WCX-10 for Adalimumab

The acidic species and other charge variants present in the adalimumabprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 10 mM SodiumPhosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphatedibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binarygradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B:22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.

Quantitation was based on the relative area percent of detected peaks.The peaks that elute at relative residence time less than a certain timeare together represented as the acidic peaks.

WCX-10 for mAb-B

The acidic species and other charge variants present in the mAb-Bprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 20 mM4-Morpholineethanesulfonic acid (MES), pH 6.5 (Mobile phase A) and 20 mM4-Morpholineethanesulfonic acid (MES), 500 mM Sodium Chloride pH 6.5(Mobile phase B). A binary gradient (87% A, 13% B: 0-5 min; 87% A, 13%B: 5-35 min; 75% A, 25% B: 35-40 min; 0% A, 100% B: 40-43 min; 87% A,13% B: 43-46 min; 87% A, 13% B: 46-55 min) was used with detection at280 nm, bw 8 nm; ref 360 nm, bw 100 nm.

Quantitation was based on the relative area percent of detected peaks.All peaks eluting prior to the Main Isoform peak were summed as theacidic region, and all peaks eluting after the LYS-2 peaks will besummed as the basic region.

WCX-10 for mAb-C

The mAb-C method was employed towards the quantification of the acidicspecies and other charge variants present mAb-C process samples. Cationexchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 20 mM4-Morpholineethanesulfonic acid (MES), pH 6.0 (Mobile phase A) and 20 mM4-Morpholineethanesulfonic acid (MES), 250 mM Sodium Chloride pH 6.0(Mobile phase B). A binary gradient (97% A, 3% B: 0-1 min; 79% A, 21% B:1-46 min; 0% A, 100% B: 46-47 min; 0% A, 100% B: 47-52 min; 97% A, 3% B:52-53 min; 97% A, 3% B: 53-60 min) was used with detection at 280 nm, bw8 nm; ref 360 nm, bw 100 nm.

Quantitation was based on the relative area percent of detected peaks.All peaks eluting prior to the Main Isoform peak will be summed as theacidic region, and all peaks eluting after the Main Isoform peak will besummed as the basic region.

Size Exclusion Chromatography

The molecular weight distribution of collected samples were quantifiedaccording to the following methods. Size exclusion chromatography (SEC)was performed using a TSK-gel G3000SW×L, 5 μm, 125 Å, 7.8×300 mm column(Tosoh Bioscience) on an HP Agilent HPLC system. Injections were madeunder isocratic elution conditions using a mobile phase of 200 mM sodiumsulfate, 100 mM sodium phosphate, pH 6.8, and detected with absorbanceat 214 nm. Quantification is based on the relative area of detectedpeaks.

Host Cell Protein (HCP) ELISA

HCP assay is based on process specific antigen based ELISA. Sampledilutions were applied to achieve readings within the calibration range.The limit of quantitation of the assay is 0.625 ng/mL.

UV spectroscopy A₂₈₀

UV A280 was used to determine protein concentrations for the samplespost Protein A elution. The assay was performed on an Agilent UVSpectrophotometer. The protein concentration was determined usingBeer-Lambert's Law, A=εlc, where A is Absorbance, E is the extinctioncoefficient, 1 is the path length, and c is the concentration. Theabsorbance was taken at 280 nm, the path length was 1 cm, and theextinction coefficients were 1.39 for adalimumab, 1.38 for mAb B, and1.43 for mAb C.

Example AEX 7.1 Determining Operating Conditions Appropriate for a Mab:Media: Buffer Combination

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   -   1. Choosing an anion concentration that allows product and        impurities to bind at a given pH above the pI of the product.    -   2. Performing a pH gradient elution covering a range above, at,        and below the pI of the product.    -   3. Determining pH range in which the protein elutes from the        anion exchange media

In this example, adalimumab and Poros 50PI were chosen. The experimentwas performed at acetate (anion) concentration of 5 mM. The column wasequilibrated with 5 mM acetate/Tris at a pH of 9.0. Adalimumab wasprepared at 5 mM acetate/Tris pH 9.0 and loaded to the column at 20g-protein/L of resin. The column was washed with 10 CVs of theequilibration buffer. A pH gradient from 9.0 to 7.0 at an anionconcentration of 5 mM acetate/Tris was then performed. The processchromatograms are shown in FIG. 165.

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   1. For a given pH, choosing a starting anion concentration that    allows product and impurities to bind to the AEX adsorbent.-   2. Loading a small amount of protein to the column and then    performing a linear gradient elution by increasing the anion    concentration keeping pH constant.-   3. Determining anion concentration range in which the protein elutes    from the anion exchange media.

In this example, adalimumab and Poros 50HQ were chosen. The experimentwas performed at a pH 8.7. The column was equilibrated with 10 mMacetate/Tris at pH 8.7. Adalimumab was prepared at 10 mM acetate/Tris pH8.7 and loaded to the column at 20 g-protein/L of resin. The column waswashed with 10 CVs of the equilibration buffer. A linear gradient from10-100 mM Acetate/Tris at pH 8.7 was performed. The processchromatograms are shown in FIG. 166.

This general approach is used to determine the appropriate operatingcondition, example shown in Table 12, for any resin/mAb combination, toimplement the invention.

TABLE 12 Example Experimental Design Scope determined from pH and aniongradient elution Poros 50HQ - 300 g/L Loading - 30 g/L Fractionation pHRange 8.2-9.0 Anion Concentration (acetate) 10-20 mM

In practicing the current invention, the acidic species reductiondesired can be achieved by appropriate pooling of the load and washfractions. By collecting and subsequently determining the productquality of each fraction throughout the load and wash, the accumulativeAR reduction and accumulative yield can be calculated using the weightedaverages up to a given fraction. Additionally, the instantaneous yieldcan be estimated by comparing the protein recovered against the totalprotein loaded to the column at a given fraction. Sample calculationsare shown below:

Sample Calculation A: Accumulative Yield up to a given fraction

${{Accumulative}\mspace{14mu}{Yield}} = \frac{{Accumulated}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}{{Total}\mspace{14mu}{Mass}\mspace{14mu}{Protein}\mspace{14mu}{Load}}$

Sample Calculation B: Accumulative AR reduction up to a given fraction

${{Accumulative}\mspace{14mu}{AR}\mspace{14mu}{Reduction}}\; = {{{Load}\mspace{14mu}{AR}\mspace{14mu}\%} - \frac{{Accumulated}\mspace{14mu}{Acidic}\mspace{14mu}{Species}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}{{Accumulated}\mspace{14mu}{Total}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}}$

Sample Calculation C: Instantaneous Yield up to a given fraction

${{Instanceous}\mspace{14mu}{Yield}} = \frac{{Accumulated}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}{{Total}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Loaded}\mspace{14mu}{to}\mspace{14mu}{Column}\mspace{14mu}{at}\mspace{14mu}{Fraction}}$

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   1. For a given pH and anion concentration and anion exchange media.-   2. Loading the anion exchange media in excess of the dynamic binding    capacity for the product for the given condition.-   3. Washing the column with a buffer containing a similar pH and    anion concentration used for the equilibration and loading steps.-   4. Collecting fractions throughout the loading and wash steps and    subsequently determining the product quality profile (e.g. AR,    aggregate, etc.).

In this example, adalimumab and Poros 50PI were chosen. The experimentwas performed at 5 mM acetate/arginine pH 8.8. The column wasequilibrated with 5 mM acetate/arginine at pH 8.8. Adalimumab wasprepared at 5 mM acetate/arginine pH 8.8 and loaded to the column at 300g-protein/L-resin. The column was washed with 20 CVs of theequilibration buffer. Fractions were collected in volumes representing30 g-protein/L-resin, shown in FIG. 167. Each fraction was then analyzedfor product quality and the accumulative yield and AR reductioncalculated, shown in Table 13. From this example, it is clear to oneskilled in the art to determine a run condition which delivers atargeted product quality and/or step yield.

This general approach is used to evaluate the performance for a givenoperating condition for any resin/mAb/buffer combination.

TABLE 13 Cumulative Yield and AR Reduction from FIG. 167 CumulativeFraction Load Yield ΔAR A2  7 g/L 0.0% 10.8%  A3*  37 g/L 0.5% 10.8% A4 67 g/L 6.7% 9.7% A5  97 g/L 16.7% 8.9% A6 127 g/L 26.9% 8.4% B1 157 g/L37.0% 7.7% B2 187 g/L 47.1% 7.1% B3 217 g/L 57.4% 6.4% B4 247 g/L 67.8%5.8% B5 277 g/L 78.0% 5.3% B6 300 g/L 84.4% 5.0% B7 Wash 87.0% 4.8% C1Wash 88.5% 4.7% C2 Wash 89.6% 4.6% *Dynamic Binding Capacity (DBC) = 39g/L

Example AEX 7.2 Demonstration of AR Reduction with AEX Adsorbents

This data set demonstrates the AR reduction achieved with threedifferent AEX adsorbents. Each resin was evaluated using adalimumab atan acetate concentration determined from the process outlined in Example7.1 and at pH values below, near, and above the pI (e.g. pH 8.5 to 9.0).Table 14 outlines the results from these experiments.

TABLE 14 Effect of AEX Resins on AR reduction of Adalimumab Resin BufferCondition Load Yield ΔAR Poros 50PI 5 mM Acetate/Tris pH 8.5 150 g/L 90%2.4% 5 mM Acetate/Tris pH 8.5 300 g/L 94% 0.9% 5 mM Acetate/Tris pH 8.7150 g/L 87% 3.6% 5 mM Acetate/Tris pH 8.7 300 g/L 94% 1.2% 5 mMAcetate/Tris pH 9.0 150 g/L 83% 3.9% 5 mM Acetate/Tris pH 9.0 300 g/L92% 1.5% Poros 50HQ 18 mM Acetate/Tris pH 8.5 250 g/L 91% 3.8% 18 mMAcetate/Tris pH 8.5 350 g/L 88% 2.2% 18 mM Acetate/Tris pH 8.7 250 g/L85% 6.0% 18 mM Acetate/Tris pH 8.7 350 g/L 84% 3.1% 18 mM Acetate/TrispH 8.9 250 g/L 67% 5.9% 18 mM Acetate/Tris pH 8.9 350 g/L 75% 3.6%CaptoDEAE 10 mM Acetate/Tris pH 8.5 150 g/L 98% 0.7% 10 mM Acetate/TrispH 8.5 300 g/L 97% 0.1% 10 mM Acetate/Tris pH 8.7 150 g/L 78% 7.1% 10 mMAcetate/Tris pH 8.7 300 g/L 95% 2.5% 10 mM Acetate/Tris pH 9.0 150 g/L29% 9.2% 10 mM Acetate/Tris pH 9.0 300 g/L 82% 5.0%

This data set is compiled to demonstrate the AR reduction achieved witheight different AEX adsorbents. Each resin was tested using an advancedscreening method using the process outlined in Example 7.1, andsubjected to four runs using adalimumab at two different pH (e.g., pH8.7 and 9.0) and two different acetate concentrations (e.g. 10 mM and 20mM). In these experiments, the instantaneous (e.g. not accumulative) ARreduction was measured by analyzing the load fraction at 150 g/L andsubsequently compared across all resins. Table 15 outlines the resultsfrom these experiments.

TABLE 15 Advanced Screen of AEX Resins for AR reduction of adalimumabInstantaneous AR Resin pH Acetate Reduction @ 150 g/L Poros 50HQ 8.7 10mM 15.0% 20 mM 10.7% 9.0 10 mM 8.6% 20 mM 13.4% Poros 50PI 8.7 10 mM6.2% 20 mM −0.1% 9.0 10 mM 6.5% 20 mM 3.0% Capto DEAE 8.7 10 mM 9.3% 20mM −0.2% 9.0 10 mM 8.6% 20 mM 7.8% Capto Q Impres 8.7 10 mM 12.3% 20 mM4.2% 9.0 10 mM 12.3% 20 mM 6.5% QAE-550C 8.7 10 mM 10.1% 20 mM 3.5% 9.010 mM 7.8% 20 mM 4.5% DEAE 650M 8.7 10 mM 5.2% 20 mM 0.1% 9.0 10 mM 6.9%20 mM −2.7% GigaCap Q 8.7 10 mM 8.1% 650M 20 mM 5.8% 9.0 10 mM 1.8% 20mM 0.4% TMAE HiCap 8.7 10 mM 4.1% 20 mM 2.8% 9.0 10 mM 1.2% 20 mM −0.1%

This data set is compiled to demonstrate the AR reduction achieved withtwo different AEX chromatographic membranes. Each membrane was testedusing conditions outlined in Table 15. The results from theseexperiments are presented in Table 16.

TABLE 16 Effect of AEX Chromatographic Membrane on AR reduction ofAdalimumab Chromatographic Membrane Equil/Wash Buffer Load Yield ΔARSartobind STIC 10 mM Acetate/Tris pH 8.7 500 g/L 94% 1.7% 20 mMAcetate/Tris pH 9.0 500 g/L 100% 0.7% Sartobind Q 20 mM Acetate/Tris pH9.0 500 g/L 100% 0.3%

This data set is compiled to demonstrate the AR reduction achieved withtwo different charged depth filters. The results from these experimentsare presented in Table 17.

TABLE 17 Effect of Charged Depth Filters on AR reduction of adalimumabDepth Filter Media Equil/Wash Buffer Load Yield ΔAR CUNO BioCap 18 mMAcetate/Tris pH 8.7 500 g/m² 92% 1.9% 25 X0HC 18 mM Acetate/Tris pH 8.7500 g/m² 84% 1.1%

Example AEX 7.3 Demonstration of AR Reduction with Other Antibodies, MabB and Mab C

AR reduction technology of the current invention has been demonstratedwith multiple antibodies using AEX adsorbents. Antibodies have differentamount charged residues and at different positions, leading to a chargeinteraction behavior on an AEX column that differs from one antibody toanother. Therefore the impact of anion type, anion concentration isdifferent for each antibody.

Table 18 and Table 19 below show the data for mAb B and mAb C. The dataclearly demonstrates that the AR reduction technology works veryeffectively for other antibodies.

TABLE 18 AR reduction for mAb B, pI ~9.1 Resin Buffer Condition pH LoadYield ΔAR Poros 50PI 5 mM Acetate/Tris 9.5 300 g/L 83% 1.1% 9.1 300 g/L94% 1.6% 8.5 300 g/L 98% <0.5% Poros 50HQ 10 mM Acetate/Tris 9.5 300 g/L69% <0.5% 9.1 300 g/L 78% 5.7% 8.5 300 g/L 81% 3.4% Capto DEAE 10 mMAcetate/Tris 9.5 300 g/L 69% 4.2% 9.1 300 g/L 82% 4.9% 8.5 300 g/L 96%<0.5%

TABLE 19 AR reduction for mAb C, pI ~7.0 Resin Buffer Condition pH LoadYield ΔAR Poros 50PI 12 mM Acetate/Tris 7.5 300 g/L 90% 2.6% 7.0 300 g/L89% 2.2% 6.5 300 g/L 87% 4.0% Poros 50HQ 45 mM Acetate/Tris 7.5 300 g/L86% 1.2% 7.0 300 g/L 88% 1.2% 6.5 300 g/L 91% 0.7% Capto DEAE 25 mMAcetate/Tris 7.5 300 g/L 79% 1.8% 7.0 300 g/L 80% 1.9% 6.5 300 g/L 89%1.8%

Example AEX 7.4 Demonstration of AR Reduction with Different pHConditions—Adalimumab

The AR species in the current invention is bound during the loadingstep; therefore the binding pH is a key variable. The anionconcentration that provides the desired performance will vary with theoperational pH.

In this example, data compiled from different experiments is shown todemonstrate the impact of the pH choice, relative to the pI of theprotein on AR reduction. This data set provides the basis for oneskilled in the art to determine a pH range to perform the experiments toimplement the current invention. Furthermore, this reiterates the factthat the pH choice depends on several factors and the relationshipbetween pH and AR reduction is also mAb dependent

In this example, adalimumab and Poros 50PI were chosen. The experimentswere performed at a concentration of 5 mM acetate/arginine at each pHspecified. Adalimumab was prepared at 5 mM acetate/arginine at each pHspecified and loaded to the column at 300 g-protein/L of resin. Thecolumn was washed with 20 CVs of the equilibration buffer. The resultsshowing the pH effect on AR reduction is shown in FIG. 168.

It is also clear that the AR reduction can be achieved with the presentinvention with a range of pH choices in the range of ±0.5 pH units fromthe pI of multiple mAbs, which are listed in Table 20. Each of theseexperiments was performed with Poros50HQ resin at a 300 g/L load with anacetate/Tris buffer system.

TABLE 20 AR reduction at pH above, at, and below protein pI Range pH-pIAntibody Yield ΔAR pH > pI 0.2 adalimumab 71% 7.0% 0.5 mAb B 69% 3.4%0.5 mAb C 86% 1.2% pH ~pI 0 adalimumab 86% 5.9% 0 mAb B 78% 5.7% 0 mAb C88% 1.2% pH < pI −0.2 adalimumab 93% 4.1% −0.5 mAb B 81% <0.5% −0.5 mAbC 91% 0.7%

Example AEX 7.5 Demonstration of AR Reduction with Different IonConcentrations—Adalimumab

Anion concentration is a key variable in the performance of anionexchange chromatography. For every combination of antibody/resin/pHthere is a range of anion concentrations that provides AR reduction; thestrategy outlined in Example 7.1 can be followed to determine the ARreduction and the corresponding recovery for each anion concentration.

Table 21 below shows the effect of anion concentration on AR reduction.The table also includes the effect of anion concentration for differentpH values. The data demonstrates that the AR reduction can beeffectively achieved over a range of anion concentrations at each pH andthat the concentration ranges depend on the pH.

TABLE 21 Effect of Anion Concentration and pH on AR reduction Resin pHBuffer Condition Load Yield ΔAR Poros 50PI 9 5 mM Acetate/Arginine 300g/L 81% 4.8% 10 mM Acetate/Arginine 227 g/L 80% 2.4% 18.5 mMAcetate/Arginine 107 g/L 88% 1.0% 8.8 5 mM Acetate/Arginine 300 g/L 93%4.5% 10 mM Acetate/Arginine 227 g/L 88% 2.5% 18.5 mM Acetate/Arginine108 g/L 96% 1.2%

Example AEX 7.6 Demonstration of AR Reduction with Different BufferSystems with Adalimumab

The anion type and concentration are key variables in Anion ExchangeChromatography. The invention has been demonstrated using Acetate andFormate as the anion type and Tris and arginine as the counter cationtype. The optimal pH and cation concentration is different for eachcation type/mixture and was derived by using the strategy outlined abovein Example 7.1. Table 22 shows the data of AR reduction andcorresponding recovery for the different anion/cation types.

TABLE 22 Effect of Anion/Cation Type AR reduction Resin Buffer ConditionLoad Yield ΔAR Poros 50PI 5 mM Acetate/Tris, pH 8.7 300 g/L 94% 1.2% 2.5mM Formate/Tris, pH 8.7 300 g/L 92% 1.3% 5 mM Acetate/Arginine, pH 8.8300 g/L 93% 4.5% Poros 50HQ 15 mM Acetate/Arginine, pH 8.7 300 g/L 89%3.2% 10 mM Formate/Tris, pH 8.7 300 g/L 83% 4.9% 18 mM Acetate/Tris, pH8.7 300 g/L 86% 5.9% Capto DEAE 10 mM Acetate/Tris, pH 8.7 300 g/L 95%2.5% 10 mM Formate/Tris, pH 8.7 300 g/L 94% 1.0% 5 mM Acetate/Arginine,pH 9.0 200 g/L 41% 7.5%

Example AEX 7.7 Demonstration of AR Reduction with Different Loading

Furthermore, the strategy outlined in Example 7.1 to reduce acidicspecies through careful control of buffer anion type, anionconcentration, AEX adsorbent, and pH can be applied to any range ofprotein loading. A range of relevant protein loadings (e.g. 100-350 g/L)for Poros 50HQ at pH 8.7 using Acetate as the anion is shown in Table23, displaying a robust AR reduction across the loading rangeinvestigated.

TABLE 23 Impact of Column loading Yield Load (100-100 mAU) ΔAR 100 g/L78% 9.7% 200 g/L 78% 4.7% 250 g/L 85% 6.0% 300 g/L 89% 3.9% 350 g/L 84%3.1%

Example AEX 7.8 Demonstration of AR Reduction with Different LoadConcentration

Furthermore, the strategy outlined in Example 7.1 to reduce acidicspecies through careful control of buffer anion type, anionconcentration, AEX adsorbent, and pH can be applied to any range ofcolumn feed streams of varying protein concentration. A range of varyingprotein load concentration for a 300 g/L load of adalimumab to Poros50HQ at 15 mM acetate/Tris pH 8.7 is shown in Table 24.

TABLE 24 Effect of Protein Load concentration Load Yield Concentration(100-100 mAU) ΔAR  5 mg/mL 90% 4.7% 10 mg/mL 86% 4.5% 15 mg/mL 85% 6.3%20 mg/mL 84% 6.2%

Example AEX 7.9 Alternative Wash Modalities

In this example, adalimumab and Poros50HQ resin were selected. In eachexperiment, variations were made in the equilibration, loading, andwashing pH values at a given acetate concentration (as specified). Table25 and Table 26 show the effect of the pH variation in the step yieldand AR reduction.

TABLE 25 Differences in pH in Equil/Wash/Load Poros 50HQ - 15 mMAcetate/Tris - pH 8.7-200 g/L Wash Yield Equilibration pH Load pH pH(100-100 mAU) ΔAR 8.7 8.7 8.5 83% 8.7% 9 8.5 8.5 89% 5.1% 9 100 g/L atpH 9.0 8.5 94% 4.5% 100 g/L at pH 8.5

TABLE 26 Differences in pH in Load/Wash Poros 50HQ - 18 mM Acetate/TrispH 8.7 Load pH Wash pH Load Yield ΔAR 8.6 8.4  75 g/L 88.8% 4.1% 8.6 8.5125 g/L 89.5% 4.2% 8.6 8.6 100 g/L 75.5% 5.3% 8.7 8.4 100 g/L 93.8% 4.1%8.7 8.5 100 g/L 81.7% 3.5% 8.7 8.5  75 g/L 94.5% 4.0% 8.7 8.6 125 g/L81.1% 5.4% 8.7 8.6  75 g/L 65.8% 6.5% 8.8 8.4 125 g/L 93.5% 3.8% 8.8 8.5100 g/L 83.7% 5.8% 8.8 8.6 100 g/L 78.4% 6.4% 8.8 8.6  75 g/L 72.7% 7.0%

As discussed in the previous sections, the operational pH and itsrelation to the product pI is important in the reduction of AR speciesin AEX. Similarly, the operational pH relative to the pKa of the AEXadsorbent is also important as many mAbs have pI similar to the pKa ofthe AEX adsorbent. This effect is shown in FIG. 187 for mAb B withseveral different AEX adsorbents, with different pKa values, run at withan acetate/Tris buffer at pH 9.1.

As described in previous sections, the AR for adalimumab is furthergrouped into two regions termed AR1 and AR2, based on a certainretention time of the peaks seen on the WCX-10 method. Thecharacteristics of the variants in these two regions are expected to bedifferent and hence the methods that reduce variants belonging to thesegroups can be specifically delineated.

Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 27. The method of the currentinvention can effectively reduce AR2 levels, as an overall decrease inAR levels is achieved. The method can be used to achieve a targetabsolute level, as exemplified by the data presented in Table 27.Multiple species are present under the group of AR2 and that the currentmethod of invention can be used to reduce such sub-species. The methodof the current invention can effectively achieve AR reduction as well asachieve a target absolute level of acidic species as exemplified by thedata presented in Table 27.

TABLE 27 AR1, AR2, and AR removal Buffer Final Final Resin Condition pHLoad Yield ΔAR1 AR1 ΔAR2 AR2 ΔAR Poros 5 mM 8.5 150 g/L 90% 0.7% 1.5%1.7% 9.4% 2.4% 50PI Acetate/Tris 300 g/L 94% 0.3% 1.9% 0.6% 10.5% 0.9%8.7 150 g/L 87% 0.9% 1.2% 2.7% 8.2% 3.6% 300 g/L 94% 0.4% 1.7% 0.8%10.1% 1.2% 8.9 150 g/L 83% 1.1% 1.4% 2.8% 8.4% 3.9% 300 g/L 92% 0.7%1.8% 0.7% 10.5% 1.5% Poros 18 mM 8.5 250 g/L 91% 2.9% 1.1% 0.9% 10.8%3.8% 50HQ Acetate/Tris 350 g/L 88% 2.7% 1.3% −0.5% 12.2% 2.2% 8.7 250g/L 88% 3.1% 0.9% 2.9% 9.0% 6.0% 350 g/L 84% 2.8% 1.2% 0.3% 11.6% 3.1%8.9 250 g/L 67% 2.6% 1.4% 3.2% 8.6% 5.9% 350 g/L 75% 2.3% 1.7% 1.3%10.5% 3.6% CaptoDEAE 10 mM 8.5 150 g/L 98% −0.1% 2.1% 0.8% 10.0% 0.7%Acetate/Tris 300 g/L 97% 0.0% 2.0% 0.1% 10.8% 0.1% 8.7 150 g/L 78% 2.4%0.8% 4.7% 6.4% 7.1% 300 g/L 95% 1.5% 1.7% 1.0% 10.1% 2.5% 8.9 150 g/L29% 2.1% 0.8% 8.0% 3.0% 10.2% 300 g/L 82% 1.7% 1.2% 3.3% 7.7% 5.0%

Example AEX 7.10 Demonstration of HCP and Aggregate Reduction inAddition to AR Reduction

AEX chromatography is effective in reducing aggregate and HCP levels. Inthe present invention, it has been demonstrated that HCP and aggregatelevels can be effectively reduced under operating conditions selectedfor AR reduction. Table 28 and Table 29 shows the aggregate and HCPremoval achieved along with AR reduction. The data clearly shows thatother process related and product related substances/impurities can beachieved using the current invention on the AEX adsorbents, and hencefunctions as an effective polishing step in the large scale purificationof monoclonal antibodies.

TABLE 28 Aggregate removal during AEX Chromatography ΔAggregate BufferCondition Load Yield Absolute Relative ΔAR 5 mM Acetate/ 300 g/L 81%0.92% 93% 4.5% Tris, pH 9.0 10 mM Acetate/ 227 g/L 80% 0.81% 88% 2.4%Tris, pH 9.0 18.5 mM Acetate/ 107 g/L 88% 0.37% 41% 1.0% Tris, pH 9.0 5mM Acetate/ 300 g/L 93% 0.91% 91% 4.5% Tris, pH 8.8 10 mM Acetate/ 227g/L 88% 0.67% 77% 2.5% Arginine, pH 8.8 18.5 mM Acetate/ 108 g/L 96%0.34% 40% 1.2% Arginine, pH 8.8

TABLE 29 HCP Removal during AEX Chromatography Poros 50PI - adalimumab -300 g/L Load Pool HCP HCP HCP Buffer Condition Yield (ng/mL) (ng/mL)(LRF) ΔAR 5 mM Acetate/Tris, pH 9.0 81% 11,617 69 2.2 4.8% 10 mMAcetate/Tris, pH 9.0 95% 83 2.1 0.8% 5 mM Acetate/Tris, pH 8.8 93%13,507 51 2.4 4.5% 10 mM Acetate/Arginine, 97% 84 2.2 1.5% pH 8.8

Example AEX 7.11 Demonstration of Means of Controlling AR Reduction

Controlling the final product quality by modifying the process based onthe quality of the intermediate material is an approach that has beenproposed as an effective way of ensuring product quality, with the viewof ensuring safety and efficacy.

Considering that the AR levels generated during cell culture and otherupstream steps can be variable, it is desirable to design a downstreamprocess step that implements a means of controlling the product quality;and to further have a specific means of controlling a process parameterto influence the quality of the product.

In the current invention, such a control is possible, as the pH and load(i.e., g/L) are parameters that can be modified to achieve a desiredseparation of the AR species. For example, to achieve a higher level ofAR reduction at a given anion concentration and pH, the load to thecolumn can be reduced. Additionally, for a given anion concentration andloading, the pH can be increased in order to achieve a higher reductionin AR species.

As an example, and not to be restrictive in any manner, it has beendemonstrated in this example that the AR levels can be controlled bychanging the pH of the load and wash solutions as well as the total loadto the column. A pilot scale Poros HQ column (10 cm diameter×22.5 cmheight, 1.8 L), was used for this study.

The load material and the stock buffer are both prepared at 18 mMAcetate/Tris the specified pH by titrating the affinity capturedmaterial with a stock Tris solution. The AR level of the load materialwas the same for both runs. This experiment demonstrates how the finalAR level can be modulated, while maintaining acceptable yields, byadjusting the pH and protein load to the column, shown in Table 30.

TABLE 30 Modulating AR Reduction using Process Analytical Technologyapproach Final Buffer Condition Load Yield ΔAR AR 18 mM Acetate/Tris, pH8.7 200 g/L 77% 5.6% 5.5% 18 mM Acetate/Tris, pH 8.5 300 g/L 89% 3.1%8.2%

Example AEX 7.12 AEX with Tris/Formate Buffer System: Acidic SpeciesReduction for Adalimumab on Poros 50HQ in a Formic Acid Buffer System

This Example provides demonstration of the use of a Tris/Formate buffersystem for AR reduction using AEX. In practicing the current Example,the acidic species reduction desired can be achieved by appropriatepooling of the load and wash fractions. By collecting and subsequentlydetermining the product quality of each fraction throughout the load andwash, the accumulative AR reduction and accumulative yield can becalculated using the weighted averages up to a given fraction.Additionally, the instantaneous yield can be estimated by comparing theprotein recovered against the total protein loaded to the column at agiven fraction.

AEX Adsorbent

Poros 50HQ (Applied Biosciences, part#1-2459-11), a rigid 50 μmpolymeric bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene], was used in this experiment.

AEX Chromatography Method

Poros 50HQ was packed in 1.0 cm×10.0 cm (OmniFit) columns. The columnwas equilibrated in a buffer system with appropriate pH andconductivity. The load was prepared in the equilibration buffer byaddition of the stock solutions to obtain the target ion concentrationsand loaded on the column, followed by washing with the equilibrationbuffer for 20 CV. The antibody product was collected in the flow-throughand wash fractions during the load and washing steps. Thecolumns/housings were then regenerated with 100 mM formate and 1M ofNaOH solution was used for column cleaning.

Sample calculations are shown below:

Sample Calculation A: Accumulative Yield up to a given fraction

${{Accumulative}\mspace{14mu}{Yield}} = \frac{{Accumulated}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}{{Total}\mspace{14mu}{Mass}\mspace{14mu}{Protein}\mspace{14mu}{Load}}$

Sample Calculation B: Accumulative AR Reduction up to a given fraction

${{Accumulative}\mspace{14mu}{AR}\mspace{14mu}{Reduction}}\; = {{{Load}\mspace{14mu}{AR}\mspace{14mu}\%} = {- \frac{{Accumulated}\mspace{14mu}{Acidic}\mspace{14mu}{Species}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}{{Accumulated}\mspace{14mu}{Total}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}}}$

Sample Calculation C: Instantaneous Yield up to a given fraction

${{Instantaneous}\mspace{14mu}{Yield}} = \frac{{Accumulated}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Recovered}\mspace{14mu}{up}\mspace{14mu}{to}\mspace{14mu}{Fraction}}{{Total}\mspace{14mu}{Protein}\mspace{14mu}{Mass}\mspace{14mu}{Loaded}\mspace{14mu}{to}\mspace{14mu}{Column}\mspace{14mu}{at}\mspace{14mu}{Fraction}}$

In this Example, adalimumab and Poros 50HQ were chosen. The experimentwas performed at 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM formate/Tris pH8.8. The column was equilibrated with the respective formate/Tris at pH8.8 for each run. Adalimumab was prepared at 10 mM, 15 mM, 20 mM, 30 mM,and 40 mM formate/Tris pH 8.8 and loaded to the column at 300-500g-protein/L-resin. Fractions were collected in volumes representing ˜25g-protein/L-resin. These fractions were analyzed for product quality,accumulative yield, and accumulative AR reduction throughout the run(shown in FIG. 189). The instantaneous yield and AR reduction at 100,200, 300, 400, and 500 g/L load are tabulated in Table 31. This exampledemonstrates the effectiveness of the Tris/Formate buffer system ingeneral and specifically the effectiveness of the Formate anion on theAEX column for AR reduction. Further it confirms that the AEX ARreduction method applies to a variety of buffer systems.

TABLE 31 Accumulative Yield and AR Reduction for a range of Formic Acidconcentrations from FIG. 189 Load 10 mM 15 mM 20 mM 30 mM 40 mM g/LYield ΔAR Yield ΔAR Yield ΔAR Yield ΔAR Yield ΔAR 100 32% 9.2% 54% 8.7%62% 8.4% 69% 5.5% 75% 4.5% 200 64% 7.4% 74% 6.8% 78% 6.0% 82% 3.2% 85%2.6% 300 75% 6.1% 82% 5.3% 85% 4.4% 84% 2.2% 86% 1.8% 400 81% 5.1% 86%4.2% 88% 3.3% — — — — 500 83% 4.5% 87% 3.6% 89% 2.8% — — — —

Example 8 Cation Exchange Chromatography Examples Example CEX 8.1Determining Operating Conditions Appropriate for a Mab: Resin: BufferCombination

The demonstration of the current invention for a specific antibody &resin is provided in this example, and consists of

-   1. Choosing a pH that is below the pI of the protein.-   2. Choosing a NaCl concentration in the range of 100 to 150 mM and    performing the experiments at, for example, 115, 125, 135    concentrations.-   3. Determining the acidic species distribution in the Flow    Through/wash fraction vs. the elution.-   4. Choosing a NaCl concentration that provides the desired acidic    species levels and recovery

In this example, adalimumab was chosen and Poros XS was chosen. Theexperiments were performed at pH 6.0. The process chromatograms areshown in FIG. 169. The recovery vs. AR reduction curves for each of theexperiments is shown in FIG. 170 and Table 32. From this set ofexperiments, a sodium concentration of 125 mM can be chosen and suchthat the recovery of the eluate is 74%, which provides an AR reductionof 5.4%. Alternately, an AR reduction value of 5.4% can be chosen whichwill provide a recovery of ˜75%.

This general approach is used to determine the appropriate operatingcondition for any resin/mAb combination, to implement the invention.

In practicing certain embodiments of the current invention, the acidicspecies reduction desired can be achieved by appropriate pooling of theelution fraction with the wash fractions. In the example described inthe previous section the elution fractions can be pooled with washfractions as shown in Table 32 to achieve AR reductions from about 1percent to about 7 percent depending on the fractions pooled. Thisapproach can be implemented to achieve a target yield and AR reductionas exemplified in FIG. 170.

TABLE 32 Wash fractions and eluate combination versus AR reductionRecovery % AR Wash Fractions (%) reduction Eluate 74 5.4 Eluate +Fraction 1 82 4.3 Eluate + Fraction 1 + Fraction 2 88 3.0 Eluate +Fraction 1 + Fraction 2 + 95 0.9 Fraction 3 Eluate + Fraction 1 +Fraction 2 + 96 0.1 Fraction 3 + Fraction 4

Example CEX 8.2 Demonstration of AR Reduction with CEX Adsorbents

This data set is compiled to demonstrate the AR reduction achieved with8 different CEX adsorbents. Conditions were derived for each resin basedon the strategy outlined in Example 8.1, above. Table 33 outlines theconditions used and the AR reduction achieved and the correspondingrecovery achieved.

The data clearly shows that the technology is robust in delivering ARreduction in all the 10 resins. As described in Example 8.1, above, theAR reduction can be balanced with recovery and an optimal condition canbe chosen. Experiments were performed at pH 7.5. 29 mM Tris-acetate wasused for pH control.

TABLE 33 Effect of CEX adsorbents on AR reduction Tris concentration %AR Resin (mM) Yield (%) Reduction Poros XS 135 103.3 0.7 140 78.6 6.8145 72.6 7.3 Poros HS 100 70.0 6.7 105 68.7 7.1 110 60.6 7.6 Capto SPImpRes 50 71.5 5.7 55 61.0 6.3 60 46.2 6.8 Nuvia S 75 67.6 10.0 80 54.310.8 85 41.0 12.2 Giga Cap CM 650 55 70.3 6.0 57.5 62.7 7.0 60 55.6 8.6Eshmuno S 65 52.7 9.0 70 35.4 11.2 75 22.7 12.2 Giga Cap S 650 65 66.38.4 70 43.6 11.1 75 31.4 12.1 CM Hyper D 45 72.2 8.9 47.5 63.2 9.9 5051.5 10.3

Example CEX 8.3 Demonstration of AR Reduction with Other Antibodies: mAbB and mAb C

AR reduction technology of the current invention has been demonstratedwith multiple antibodies using CEX Adsorbents. Antibodies have differentamounts of charged residues and at different positions, leading to acharge interaction behavior on a CEX column that differs from oneantibody to another. Therefore the impact of cation type, cationconcentration is different for each antibody.

For each antibody/resin combination, the experimental strategy outlinedin Example 8.1, above, was employed to determine the cationconcentration for each cation type that provided AR reduction.

Table 34 and Table 35 below shows the data for mAb B and mAb C. The dataclearly demonstrates that the AR reduction technology works veryeffectively for other antibodies. It is also clear that theconcentration ranges are different between different antibodies. The pHrange chosen was related to the isoelectric point of the antibody andwas chosen to be approximately 1 to 2 units less than the pI of themolecule.

TABLE 34 AR reduction for mAb B Buffer Concentration Yield % AR ResinSystem (mM) pH (%) Reduction Poros XS Tris 120 7.5 57.2 8.4 Acetate 12546.5 9.3 130 37.1 10.3 Nuvia S 85 72.5 16.6 90 56.1 16.9 95 44.2 17 CMHyper D 50 73 8.2 55 62 9.2 60 52.6 9.2

TABLE 35 AR reduction for mAb C Buffer Concentration Yield Load % ARResin System (mM) pH (%) % AR Reduction Poros XS Tris 40 6.0 87.4 15.68.5 Acetate 45 56.8 15.7 12.8 50 31.3 15.7 14.3 Nuvia S 35 45.1 11.511.2 37 28.5 15.4 15.2 40 15.3 15.2 15.2 CM 18 83.6 16.3 6.3 Hyper D 2064.9 16.3 11.2 22 50.7 16.4 12.3

Example CEX 8.4 Demonstration of AR Reduction with Different pHConditions—Adalimumab

The AR species in the current invention is removed in the Flowthrough/Wash fraction. Therefore the binding pH is a key variable. Thecation concentration that provides the desired performance will varywith the binding pH. Therefore for each binding pH, the experimentalstrategy outlined in Example 8.1, above, is carried out to determine therange of ion concentration that results in AR reduction.

The results of the experiments with different pHs for adalimumab isshown in Table 36. As can be seen, at lower pH, the cation concentrationrequired to achieve AR removal in the wash fraction is higher. It isunexpected that the AR reduction is significantly more robust andoptimal at higher pHs (closer to pI) than at lower pHs. It is notobvious to one skilled in the art to operate a cation exchangechromatography at pH closer to pI as shown in Table 37. Literature datasuggests an optimal pH of at least 3 units less than the pI of themolecule.

TABLE 36 Effect of pH on AR reduction Buffer Buffer Concentration % ARpH Resin System (mM) Yield (%) Reduction 5.5 Poros XS Tris Acetate 35058.2 5.9 6.5 225 61.4 6.4 7 170 75.3 5.6 7.5 140 78.6 6.8 8 125 75.8 5.77.5 CM Hyper D Ammonium 4 77.9 7.4 Sulfate 6 Sodium 45 86.1 4 6.8Chloride 30 71.5 7 7.5 10 71.3 6.8 7.5 Tris Acetate 45 72.2 8.9

TABLE 37 Effect of delta pH and pI on AR reduction % AR pI- Buffer[Cation] Yield Reduc- pH Antibody Resin system (mM) (%) tion 1.1adalimumab Poros XS Arginine/ 60/29 58.9 7.8 Tris Acetate 2.2 Sodium 12573.5 5.4 1.8 Chloride 75 90 1.5 1.1 50 72.1 7.2 3.1 Tris Acetate 35058.2 5.9 2.1 225 61.4 6.4 1.6 170 75.3 5.6 1.1 145 72.6 7.3 0.6 125 75.85.7 1.6 mAb B Poros XS Tris Acetate 120 57.2 8.4 1.6 CM Hyper D TrisAcetate 50 73 8.2 1.6 Nuvia S Tris Acetate 85 72.5 8.4 1.0 mAb C PorosXS Tris Acetate 40 87.4 8.5 1.0 CM Hyper D Tris Acetate 18 83.6 6.3 1.0Nuvia S Tris Acetate 35 45.1 11.2

Example CEX 8.5 Demonstration of AR Reduction with Different IonConcentrations—Adalimumab

Cation concentration is a key variable in the performance of cationexchange chromatography. For every combination of antibody/resin/pHthere is a range of cation concentrations that provides AR reduction;the strategy outlined in Example 8.1, above, can be followed todetermine the AR reduction and the corresponding recovery for eachcation concentration.

Table 38 below shows the effect of cation concentration on AR reduction.The table also includes the effect of cation concentration for differentpH values. The data demonstrates that the AR reduction can beeffectively achieved over a range of cation concentrations at each pHand that the concentration ranges depend on the pH. The table alsoincludes an example of the concentration range for a different cationtype.

TABLE 38 Effect of cation concentration and pH on AR reduction Cationconcentration Yield % AR (mM) Buffer system pH Resin (%) Reduction 60/29Arginine/Tris 7.5 Poros XS 58.9 7.8 65/29 Acetae 47.4 8.7 23 80.5 5.8 2572.9 7.3 27 52.2 9.5 115 Sodium 6 85.4 4.2 125 Chloride 73.5 5.4 13048.7 7.1 75 6.8 90 1.5 90 53.7 2.1 45 7.5 60.7 7.9 50 72.1 7.2 350 TrisAcetate 5.5 58.2 5.9 375 38.4 7.4 400 29.9 6.2 225 6.5 61.4 6.4 250 59.56.6 275 37.6 7.8 300 21.6 8.8 165 7 83.8 4.3 170 75.3 5.6 175 70.3 5.7140 7.5 78.6 6.8 145 72.6 7.3 150 69.2 7.8 175 29.8 10.3 125 8 75.8 5.7130 67.7 6.5 135 57.4 7.5

Example CEX 8.6 Demonstration of AR Reduction with Different BufferSystems with Adalimumab

The cation type and concentration are key variables in Cation ExchangeChromatography. The invention has been demonstrated with Tris,Sodium/Tris, Ammonium/Tris and Arginine/Tris as cation types/mixtureswith effective reduction of AR in each case. As one skilled in the artwould appreciate the optimal pH and cation concentration is differentfor each cation type/mixture and was derived by using the strategyoutlined in Example 8.1, above. Experiment were performed at pH 7.5. 29mM Tris-acetate was used for pH control. Table 39 shows the data of ARreduction and corresponding recovery for the different cationtypes/mixtures.

TABLE 39 Effect of cation types/mixtures on AR reduction Cationconcentration Yield % AR Buffer System Resin (mM) pH (%) ReductionArginine/Tris Poros XS 60 7.5 58.9 7.8 acetate Ammonium 25 72.9 7.3Sulfate Sodium 50 72.1 7.2 Chloride Tris Acetate 140 78.6 6.8 AmmoniumCM 4 77.9 7.4 Sulfate Hyper D Sodium 10 71.3 6.8 Chloride Tris Acetate45 72.2 8.9 Ammonium Nuvia S 11 66.6 12.6 Sulfate Sodium 20 75.9 10.5Chloride Tris Acetate 75 67.6 10

Example CEX 8.7 Demonstration of AR Reduction with Different Loading

Furthermore, the strategy outlined in Example 8.1, above, to reduceacidic species through careful control of buffer cation type,concentration and pH can be applied to any range of protein loadingwhich represents an operational mode of binding followed by elution,i.e. not overloaded or a column load factor below that of the adsorbentsbinding capacity. A range of relevant protein loadings for Poros XS atpH 7.5 using Tris as the cation is shown in Table 40 showing robust ARreduction.

TABLE 40 Impact of Column loading Column Loading (g product/L BufferConcentration Yield % AR resin) System (mM) pH (%) Reduction 25 Tris 1607.5 83.6 6.4 30 155 79.4 6.0 35 140 87.4 4.8 38 140 83.5 5.0 40 140 76.46.0 42 140 74.5 5.7 45 140 67.0 6.6

Example CEX 8.8 Demonstration of AR Reduction with Different LoadConcentration

Furthermore, the strategy outlined in Example 8.1, above, to reduceacidic species through careful control of buffer cation type,concentration and pH can be applied to any range of column feed streamsof varying protein concentration. A range of varying protein loadconcentration for Poros XS at pH 7.5 using Tris as the cation is shownin Table 41 showing robust AR reduction.

TABLE 41 Effect of Protein Load concentration Load Concen- % ARConcentration Buffer tration Yield Reduc- (mg/mL) Resin System (mM) pH(%) tion 3 Poros XS Tris 140 7.5 77.3 7 4 Acetate 145 60.7 7 5 140 78.76.7 5 145 64.1 7 6 145 59.5 6.9 7 140 77.6 6.5

As described above, the AR for adalimumab is further grouped into tworegions termed AR1 and AR2, based on a certain retention time of thepeaks seen on the WCX-10 method. The characteristics of the variants inthese two regions are expected to be different and hence the methodsthat reduce variants belonging to these groups can be specificallydelineated.

Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 42.

The specific species comprising the AR1 species can be identified andquantitated, to demonstrate reduction of such species by methods of thecurrent invention. Two of such species, glycated mAb, and MGO modifiedmAb have been identified and shown to be reduced by the methods of thisinvention. While these species are among the acidic species part of thecharge variants, the acidic species typically described in theliterature is the deamidated mAb, which is distinctly different.

TABLE 42 The final impurity level Cation Conc. Yield % Final % FinalBuffer System (mM) pH (%) AR1 AR Arginine/Tris 60 7.5 58.9 0.3 5.8Acetate 65 7.5 47.4 0.3 4.7 Ammonium 23 7.5 80.5 0.6 8.3 Sulfate 25 7.572.9 0 6.4 27 7.5 52.2 0.4 5.0 Sodium Chloride 115 6 85.4 1.3 10.2 125 673.5 0 8.1 135 6 48.7 0 6.1 75 6.8 90 1.4 10.9 90 6.8 53.7 0.7 11.2 457.5 60.7 0 6.2 50 7.5 72.1 0 7.8 Tris Acetate 350 5.5 58.2 0 7.7 375 5.538.4 0.1 6.2 400 5.5 29.9 1.5 7.3 225 6.5 61.4 0.8 7.2 250 6.5 59.5 06.8 275 6.5 37.6 0 5.6 300 6.5 21.6 0 4.7

The method of the current invention can effectively reduce AR2 levels,as an overall decrease in AR levels is achieved. The method can be usedto achieve a target absolute level, as exemplified by the data presentedin Table 42.

The method of the current invention can effectively achieve AR reductionas well as achieve a target absolute level of acidic species asexemplified by the data presented in Table 42.

Example CEX 8.9 Demonstration of Glycated and Methylglyoxylated SpeciesReduction

The strategy outlined in Example 8.1, above, to reduce acidic speciesthrough careful control of buffer cation type, concentration and pH canbe further extended to specific post-translational modifications. Whileacidic species are defined in the application as impurities that areless retained than the main peak on an analytical weak cation exchange(WCX) HPLC column, specific known product related substances derivedfrom cellular metabolism modification such as glycation andmethylglyoxal (MGO) can be specifically identified as being part of theacidic species. FIG. 171 and FIG. 172 shows the outcome of in-vitrolabeling experiments which demonstrate that glycation and MGO modifiedantibody are unique species that are resolved by the WCX method in theAR1 region of the chromatogram and can be enriched in vitro.Furthermore, the invention described here shows that glycated and MGOmodified antibody can be effectively removed through the careful controlof buffer cation type, concentration and pH using the CEX as describedin Example 8.1, above. Quantitative reduction of AR1 and hence theglycated and MGO species by CEX and CEX-Mixed Mode resins is show inTable 43 and Table 44.

TABLE 43 Glycated species removal % AR1 % AR Buffer Conc. Yield Load %Load % Reduc- Reduc- Resin System (mM) pH (%) AR1 AR tion tion PorosTris 135 7.5 54.0 40.8 58.6 30.8 34.8 XS

TABLE 44 MGO peak removal Concen- % AR1 % AR Buffer tration Yield Reduc-Reduc- Resin System (mM) pH (%) tion tion Toyo Pearl MX Tris 80 7.5 66.72.8 7.2 TRP 650M Poros XS 145 64.1 2.7 7 Nuvia S 90 48.5 3.1 9.6

Example CEX 8.10 Demonstration of Lysine Distribution Modification

The strategy outlined in Example 8.1, above, to reduce acidic speciesalso can be used to modulate the distribution of C-terminal Lys variantsof monoclonal antibodies, a known post-translational modificationleading to charge heterogeneity. Some minor changes in the distributionof Lys isoforms is expected through the reduction of acidic species asthe WCX analysis is a compositional analysis. However, through carefulcontrol of buffer cation type, concentration and pH care, in addition toreducing acidic species, the elution pool can be enriched for the morebasic isoforms (Lys 1 and Lys2). Table 45 and FIG. 173 depicts anon-limited example of the impact of pH and cation (Tris) concentrationon basic isoform enrichment.

TABLE 45 Change in Lysine distribution during CEX Chromatography -impact of Tris concentration Buffer % LYS0 % LYS1 % LYS2 BufferConcentration decrease Increase Increase System (mM) pH 1.6 4.4 2.7 Tris350 5.5 5 6.5 5.5 Acetate 375 9.7 7.5 11.9 400 1.9 5 2.9 225 6.5 1.9 5.33 250 6.1 7.4 6 275 11.8 8.6 10.8 300 0.2 5.2 1.6 140 7.5 0.6 5.7 1.8145 1.8 6.8 2.4 150 16.4 14.9 10.3 175

Example CEX 8.11 Demonstration of HCP and Aggregate Reduction inAddition to AR Reduction

In the present invention, it has been demonstrated that HCP andaggregate levels can be effectively reduced by appropriate adjustment ofthe elution conditions, after washing off the AR enriched species in theFlow Through/wash fractions.

Table 46 and Table 47 shows the HCP and aggregate removal achieved alongwith AR reduction. The data clearly shows that other process relatedimpurities and product related substances can be achieved using thecurrent invention on the CEX adsorbents, and hence functions as aneffective polishing step in the large scale purification of monoclonalantibodies.

TABLE 46 Aggregate removal during CEX Chromatography % % % Aggre- Frag-Mono- Buffer gate Re- ment Re- mer In- Resin Antibody system pH ductionduction crease CM adalimumab 5 mM 7.5 0.04 0.17 0.2 Hyper Ammonium DSulfate 45 mM Tris 0.01 0.18 0.19 Acetate Nuvia S 11.5 mM 0.16 0.17 0.33Ammonium Sulfate 75 mM Tris 0.09 0.11 0.2 Acetate 22.5 mM 0.08 0.19 0.27Sodium Chloride Poros 27 mM 0.75 0.27 1.02 XS Ammonium Sulfate 140 mMTris 0.51 0.41 0.92 Acetate 145 mM Tris 0.58 0.41 0.98 Acetate Nuvia SmAb B 85 mM Tris 0.19 0.27 0.47 Acetate Poros 130 mM Tris 0.36 0.04 0.39XS Acetate Nuvia S mAb C 35 mM Tris 6.0 0.07 0.01 0.07 Acetate Poros 50mM Tris 0.27 0 0.28 XS Acetate

TABLE 47 HCP Removal during CEX Chromatography Eluate Load Pool Reduc-Buffer HCP HCP tion Resin Antibody system pH (ng/mg) (ng/mg) fold CMadalimumab 5 mM 7.5 8105 3844 2.1 Hyper D Ammonium Sulfate 45 mM Tris8628 5615 1.5 Nuvia S 11.5 mM 5314 2405 2.2 Ammonium Sulfate 75 mM Tris17317 12845 1.4 Acetate 22.5 mM 9091 4115 2.2 Sodium Chloride Poros 27mM 21857 12574 1.0 XS Ammonium Sulfate 140 mM 14732 9181 1.7 TrisAcetate 145 mM 15359 10113 1.6 Tris Acetate Nuvia S mAb B 85 mM Tris 735319 2.3 Acetate Poros 130 mM 2183 404 5.4 XS Tris Acetate Nuvia S mAb C35 mM Tris 6.0 27 31 0.9 Acetate Poros 50 mM Tris 25 15 1.7 XS Acetate

Example CEX 8.12 Demonstration of Means of Controlling AR Reduction

Controlling the final product quality by modifying the process based onthe quality of the intermediate material is an approach that has beenproposed as an effective way of ensuring product quality, with the viewof ensuring safety and efficacy.

Considering that the AR levels generated during cell culture and otherupstream steps can be variable, it is desirable to design a downstreamprocess step that implements a means of controlling the product qualityand to further have a specific means of controlling a process parameterto influence the quality of the product.

In the current invention, such a control is possible, as the cationconcentration is a single parameter that can be modified to achieve adesired separation of the AR species. For example, to achieve a higherlevel of AR reduction, the Tris concentration of the loading materialand the wash buffer can be decreased, such that the AR enriched speciesis collected in the Flow Through fraction.

As an example, and not to be restrictive in any manner, it has beendemonstrated in this example that the AR levels can be controlled bychanging the Tris concentration of the load and wash solutions. A pilotscale Poros XS column (10 cm diameter×22 cm height, 1.7 L), was used forthis study.

The load material and the stock buffer are both prepared at 300 mM Trisconcentration at the same pH. The AR level of the load material wasmeasured to be X %. The load material and equilibration/wash buffer arein-line diluted to the target Tris concentration based on predeterminedcorrelation between the AR levels and Tris concentration. Asdemonstrated in the example, when the Tris concentration was adjusted to156 mM, a final AR reduction of 4.1% was achieved, whereas when the Trisconcentration was adjusted to 150 mM, a final AR level of 3.1 wasachieved (Table 48). This allows very predictable control of the ARlevels ensuring achievement of the desired product quality.

TABLE 48 Controlling AR Reduction using Process Analytical Technologyapproach % AR Tris conc (mM) Yield (%) Reduction 156 51.9 4.1 150 70.53.1 131 95.3 1.3

In addition to the acidic species reduction demonstrated in Example CEX8.1 through careful control of the pH cation type and concentration inthe load (process stream) and equilibration/wash buffers, thecomposition of the elution buffer can also be used to further improvethe product quality profiles. The impact of various cation types,concentration and pH were tested for eluting the product. There is awide selection for elution buffer as shown in Table 49. The experimentswere performed using Poros XS resin.

TABLE 49 Elution buffer types on aggregates removal % Aggregate BufferSystem pH Yield (%) Reduction 200 mM Sodium Sulfate/29 mM Tris 5.2 76.10.36 Acetate 160 mM Sodium Sulfate/29 mM Tris 5.2 82.3 0.82 Acetate 150MSodium Sulfate/29 mM Tris 5.2 78.8 0.90 Acetate 140M Sodium Sulfate/29mM Tris 5.2 78.2 1.00 Acetate 400 mM Sodium Sulfate/29 mM Tris 4.0 78.50.98 Acetate 100 mM Sodium Sulfate/140 mM Tris 5.2 70.9 1.25 Acetate 150mM Sodium Sulfate/140 mM Tris 5.2 79.6 1.05 Acetate 140M SodiumSulfate/140 mM Tris 5.2 75.4 1.07 Acetate 130 mM Sodium Sulfate/140 mMTris 5.2 78.2 1.07 Acetate 300 mM Sodium Sulfate/30 mM Tris 4.6 80.30.57 Acetate 150 mM Sodium Sulfate/29 mM Tris 7.5 75.0 0.92 Acetate

Example CEX 8.13 Demonstration of AR Reduction with Cation—HIC MixedMode Resin

The strategy outlined in Example 8.1, above, to reduce acidic speciesthrough careful control of buffer cation type, concentration and pH canbe expanded to include other chromatography adsorbents such as mixedmode or multi-modal absorbents which include a cation exchangemechanism. Table 50 outlines the conditions used and the AR reductionachieved for two cation-hydrophobic interaction mixed mode resins. Thedata clearly shows that the technology outlined in Example 8.1 is robustin delivering AR reduction for these types of resins across in additionto traditional cation exchange adsorbents. As described in Example 8.1,the AR reduction can be balanced with recovery and an optimal conditioncan be chosen. As a further demonstration, mAb 2 was also evaluated(Table 51) with the same outcome showing the same relationship betweencation concentration, recovery and AR reduction. As previously shown inExample 8.1, the optimal condition for different molecules varies.Furthermore, this technology when applied to CEX-HIC mixed mode resinsalso shows reduction of impurities as previously described.

TABLE 50 Adalimumab AR Reduction by Cation Exchange Mixed ModeChromatography Tris Buffer Concentration Yield % AR Resin System (mM) pH(%) Reduction Nuvia C Prime Tris Acetate 70 7.5 63.8 6.5 72.5 7.5 61.16.0 75 7.5 57.1 6.7 Toyo Pearl MX 75 7.5 80 5.7 Trp 650M 80 7.5 66.7 7.285 7.5 51.8 8.6

TABLE 51 mAb B AR Reduction by Cation Exchange Mixed Mode ChromatographyBuffer Concentration Yield % AR Resin System (mM) pH (%) Reduction NuviaC Prime Tris Acetate 75 7.5 86.0 2.0 85 7.5 74.6 5.9 95 7.5 61.3 6.8Toyo Pearl MX 90 7.5 81.1 6.4 Trp 650M 95 7.5 68.8 8.8 100 7.5 53.5 10.7

As described in previous sections, the AR for adalimumab is furthergrouped into two regions termed AR1 and AR2, based on a certainretention time of the peaks seen on the WCX-10 method. Thecharacteristics of the variants in these two regions are expected to bedifferent and hence the methods that reduce variants belonging to thesegroups can be specifically delineated.

Further, in addition to achieving a certain AR reduction, it may bedesirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 52A with Tables 52B and 52Cindicating the levels of additional process-related impurities or acidicspecies.

The specific species comprising the AR1 species can be identified andquantitated, to demonstrate reduction of such species by methods of thecurrent invention. While these species are among the acidic species partof the charge variants, the acidic species typically described in theliterature is the deamidated mAb, which is distinctly different. Theseresults show that the Cation Exchange Resin with additional pendanthydrophobic interaction functionality, is able to provide AR reductioneffectively, similar to the CEX Adsorbents.

TABLE 52A Final acidic species level for adalimumab Tris Concen- Buffertration Yield Final Final Final Resin System (mM) pH (%) % AR1 % AR2 %AR Nuvia C Tris 70 7.5 63.8 0.39 4.64 5.03 Prime Acetate 72.5 7.5 61.10.36 4.4 4.75 75 7.5 63.8 0.39 4.06 4.45 Toyo Pearl 75 7.5 80 0.6 4.24.8 MX Trp 80 7.5 66.7 0.5 3.2 3.7 650M 85 7.5 51.8 0.2 2.2 2.4

TABLE 52B Aggregates/Fragments Reduction by Cation Exchange Mixed ModeChromatography % Buffer % Aggregate % Fragment Monomer Resin AntibodySystem pH Reduction Reduction Increase Nuvia C prime adalimumab 70 mMTris 7.5 0.3 0.34 0.63 Toyo Pearl MX 75 mM Tris 0.08 0.56 0.65 Trp 650MNuvia C prime mAb B 85 mM Tris 0.87 1.18 2.04 Toyo Pearl MX 95 mM Tris0.0 1.8 1.8 Trp 650M

TABLE 52C HCP Reduction by Cation Exchange Mixed Mode ChromatographyLoad Eluate HCP pool HCP Fold Resin Antibody Buffer pH (ng/mg) (ng/mg)Reduction Toyo Pearl MX Trp adalimumab 70 mM Tris 7.5 202.6 38.9 5.2650M Nuvia C prime 75 mM Tris 205.5 72.8 2.8 Toyo Pearl MX Trp mAb B 95mM Tris 983.3 137.1 7.2 650M Nuvia C prime 85 mM Tris 1011.3 88.2 11.5

Example 8.14 CEX with Tris/Formate Buffer System: AR Reduction withDifferent Tris/Formate Concentrations—Adalimumab

This Example provides a demonstration of AR reduction using aTris/Formate buffer system and CEX. Cation (e.g. Tris) concentration isa key variable in the performance of cation exchange chromatography.

CEX Adsorbent:

Poros XS (Applied Biosciences, part#4404338), a rigid 50 μm polymericbead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene], was used in this experiment.

CEX Chromatography Method

Poros XS was packed in 1.0 cm×10.0 cm (OmniFit) columns. The column wasequilibrated in a buffer system with appropriate pH and conductivity.The column load was prepared in the equilibration buffer by bufferexchange or addition of the stock solutions to obtain the target ionconcentrations as specified and loaded on the column at approximately 40g protein/L resin (or as specified) followed by washing with theequilibration buffer for 20 CV (or as specified). The antibody productwas then eluted, and the column regenerated.

In this Example, adalimumab and Poros XS were chosen. The experiment wasperformed at Tris concentrations of 120-150 mM buffered to pH 7.5 withformic acid. The column was equilibrated with the respectiveTris/Formate at pH 7.5 for each run. Adalimumab was prepared at therespective Tris/Formate pH 7.5 and loaded to the column at 35-45g-protein/L-resin. The column was then washed with 20 CVs with theequilibration buffer, and then eluted with a 140 mM Tris/Formate+140 mMSodium Sulfate buffer at pH 5.2. The eluate was analyzed for productquality and yield.

Table 53, below, shows the effect of Tris concentration on AR reductionand aggregate reduction for adalimumab on Poros XS in a Tris/Formatebuffer system at a pH of 7.5. The data demonstrates that AR andaggregate reduction can be effectively achieved over a range of Trisconcentrations and column loadings. This example demonstrates theeffectiveness of the Tris/Formate buffer system in general andspecifically the effectiveness of the Tris cation in the context of theFormate on the CEX column for AR reduction. Further, it confirms thatthe CEX AR reduction method applies to a variety of buffer systems.

TABLE 53 Effect of Tris concentration at pH 7.5 on AR and aggregatereduction Tris Loading AR AR1 Final AR2 Final Aggregate Concentrationg/L Yield Reduction Reduction AR1 Reduction AR2 Reduction 120 mM 35 96%0.5% 0.4% 0.1% 0.1% 4.2% 0.1% 40 88% 1.8% 0.6% 0.0% 1.3% 2.9% 0.2% 125mM 35 90% 2.4% 0.5% 0.1% 1.8% 3.0% 0.2% 40 78% 3.0% 0.6% 0.0% 2.4% 2.4%0.2% 130 mM 35 76% 3.1% 2.3% 0.8% 1.0% 8.5% 1.3% 40 64% 4.0% 2.5% 0.4%2.7% 6.9% 1.4% 45 70% 4.0% 2.4% 0.3% 3.6% 6.0% 1.5% 135 mM 35 78% 5.6%0.8% 0.0% 4.8% 2.8% 0.3% 40 58% 5.3% 0.8% 0.0% 4.4% 3.1% 0.2% 140 mM 3563% 6.1% 2.5% 0.3% 3.5% 6.4% 1.3% 40 55% 6.0% 2.4% 0.3% 4.3% 5.3% 1.3%45 55% 5.8% 2.4% 0.3% 4.8% 4.9% 1.0% 145 mM 35 55% 4.1% 0.6% 0.0% 3.5%1.7% 0.3% 40 44% 4.2% 0.6% 0.0% 3.6% 1.6% 0.3% 150 mM 35 50% 7.4% 2.4%0.2% 5.0% 4.5% 1.1% 40 44% 7.4% 2.6% 0.3% 5.5% 4.3% 0.7% 45 40% 6.9%2.5% 0.2% 5.7% 4.1% 0.5%

Wash Volumes for CEX Chromatography

The experiments were performed using Protein A eluate as CEX loadingmaterial. Run 1 was performed under the load/wash buffer conditions of128 mM Tris-formate buffer system, pH 7.5, 40 g/L loading. Wash wasperformed with 20 CV of the wash buffer. Run 2 was performed under theload/wash buffer conditions of 160 mM Tris-formate buffer system, pH7.5, 40 g/L loading. Wash was performed with 6 CV of the wash buffer.

As shown in Table 54, Run 1 and Run 2 gave similar yield and ARreduction. Therefore, these results demonstrate that process performancecan be achieved by varying the wash volume with corresponding loadingconditions.

TABLE 54 Wash volume effect on AR reduction and yield Product qualityRun 1 (20 CV Wash Run 2 (6 CV Wash) % Yield 92.1 89.6 % Load AR1 3.443.43 % Eluate AR1 1.43 1.21 % AR1 Reduction 2.01 2.22 % Load AR2 10.449.87 % Eluate AR2 9.25 8.86 % AR2 reduction 1.19 1.01 % Total load AR13.88 13.3 % Total eluate AR 10.68 10.07 % total AR reduction 3.20 3.23

Example 9 Mixed Mode Chromatography Examples Example MM 9.1 Resin and pHCombination

In this Example one of the approaches outlined in the generaldescription was employed to determine the operating conditions toimplement the invention. Specifically, a response surface design DOE wasapplied to evaluate mAb AR reductions and recovery yields.

The demonstration of the current invention for a specific antibody &resin is provided in this Example, and consists of

-   -   1. Choosing a pH in the range of 6.8 to 8.4.    -   2. Choosing a conductivity in the range of 2.3 to 13.7 mS/cm.    -   3. Determining the acidic species distribution in the Flow        Through/wash fractions.    -   4. Choosing an optimal pH and conductivity that provides the        desired acidic species levels and recovery

In this example, adalimumab and resin Capto Adhere were chosen. Theexperiments were performed with Tris/Acetate buffer system at target pHand conductivity listed in Table 55. The load material was from ProteinA affinity capture and pH adjusted. This study demonstrated the effectof loading pH and conductivity on acidic species reduction. The acidicspecies reduction can be significantly affected by operating pH. ARreduction increased with increasing pH and/or decreasing conductivity(Table 55, Table 56 and FIG. 174)

TABLE 55 DOE study condition Tris Acetate Edge points for ResponseBuffer Range Surface pH 7.0-8.2  6.8, 8.4 Conductivity 4.0-12.0 2.3,13.7

TABLE 56 DOE Study Operating Conditions and Results DOE exp pHConductivity (mS/cm) ΔAR (%) Yield (%) 1 7.0 4.0 0.4 83 2 7.6 8.0 0.4 733 7.6 2.3 1.3 82 4 7.6 8.0 0.6 68 5 7.6 8.0 0.2 70 6 7.6 8.0 −0.2 69 78.2 4.0 2.1 67 8 7.6 8.0 1.3 69 9 7.0 12.0 −0.2 70 10 7.6 8.0 1.2 71 118.2 12.0 1.4 74 12 6.8 8.0 1.2 76 13 8.4 8.0 1.8 67 14 7.6 8.0 1.4 71 157.6 13.7 1.0 74 16 7.6 8.0 1.6 70 Note: AR reductions and proteinrecovery yields were calculated based on the Flow Through fractions atabout loading 200 g protein per L of resin.

Example MM 9.2 Fraction Pooling

In this example, adalimumab and resin Capto Adhere were chosen. Theexperiments were performed with Tris/Acetate buffer system at pH 7.85and conductivity of 2.5 mS/cm. The load material was from Protein Aaffinity capture and pH adjusted. Column Flow Through was fractionatedthroughout the entire load and wash phases. Each fraction was analyzedfor acidic species and protein recovery. FIG. 175, FIG. 176 and Table 57demonstrate AR reduction achieved with the corresponding recovery. TheseAR reductions and recoveries correspond to the cumulative pools of thefractions from the start to the various points during the load/wash.This is depicted in Table 57 where the AR reductions corresponding toeach of these pools. This data is plotted in FIG. 175.

TABLE 57 Cumulative AR reduction in Flow Through/wash fractions FlowThrough Fraction Yield Δ AR1 Δ AR2 Δ AR ΔLys (Load & wash) (%) (%) (%)(%) (%) A2 23 2.56 3.13 5.69 5.61 A2 + A3 45 2.31 2.19 4.49 4.37 A2 +A3 + A4 58 1.83 1.89 3.72 3.63 A2 + A3 + A4 + A5 65 1.57 1.58 3.15 3.06A2 + A3 + A4 + A5 + A6 73 1.38 1.32 2.70 2.61 A2 + A3 + A4 + A5 + A6 +B7 86 1.26 1.12 2.38 2.30 A2 + A3 + A4 + A5 + A6 + 89 1.19 0.91 2.092.02 B7 + B6 A2 + A3 + A4 + A5 + A6 + 90 1.14 0.82 1.96 1.89 B7 + B6 +B5 Note: “A” Fractions are load fractions and “B” Fractions are washfractions

Example MM 9.3 Demonstration of AR Reduction with Mixed Mode Adsorbents

In this Example, adalimumab was chosen. The experiments were performedwith Tris/Acetate buffer system at pH 7.85 and conductivity of 2.5, 3.5,and 4.5 mS/cm. The same load material was applied to different mixedmode resin columns. The load material was from Protein A affinitycapture and pH adjusted. Table 58 shows that all three mixed mode resinscould reduce mAb acidic species. Due to the differences of resinligands, the AR reduction level may slightly vary under certainconditions.

TABLE 58 Adalimumab AR Reduction and Protein Recovery Yields Processedwith Different Mixed Mode media Tris/Ac Buffer Capto Adhere HEA PPAOperating pH 7.85 pH 7.85 pH 7.85 Conditions 4.5 mS/cm 3.5 mS/cm 2.5mS/cm 4.5 mS/cm 3.5 mS/cm 2.5 mS/cm 4.5 mS/cm 3.5 mS/cm 2.5 mS/cm Yield(%) 50 52 58 49 52 56 40 43 47 AR 1.8 3.8 3.7 1.1 2.7 3.2 1.4 2.2 3.5Reduction (%) Yield (%) 68 71 73 65 75 69 61 64 63 AR 1.1 2.7 2.7 0.51.8 2.1 0.4 1.9 2.6 Reduction (%)

Example MM 9.4 Demonstration of AR Reduction with Other Antibodies: mAbB and mAb C

In this Example, another two different monoclonal antibodies besidesadalimumab (mAb B and mAb C) and resin Capto Adhere was chosen. Theexperiments were performed with Tris/Acetate buffer system at multiplepH and conductivity condition. The load materials of all mAbs were fromProtein A affinity capture and pH adjusted. mAb C was also applied toanother two MM resins besides Capto Adhere under the same operatingconditions. Table 59 outlines the operating conditions and the ARreduction achieved and the corresponding recovery achieved. The resultsdemonstrate that the technology can also reduce acidic species for othermonoclonal antibodies with optimal pH and conductivity conditions.Experiments were performed with Tris-acetate buffer system.

TABLE 59 AR Reductions and Protein Recovery for different mAb with CaptoAdhere columns conductivity ΔAR Yield mAb pH (mS/cm) (%) (%) adalimumab7.85 3.5 3.8 52 7.85 2.5 3.7 58 mAb B 6.8 3.0 6.3 51 6.8 4.5 4.2 53 7.03.0 5.1 77 8.0 3.0 3.4 60 mAb C 9.0 3.0 5.3 73 8.5 3.0 3.5 54 8.0 3.03.7 50

FIG. 177 displays the mAb B cumulative pool AR broke through the columnof Capto Adhere operated at pH 7.0 and conductivity of 3.0 mS/cm withTris-Acetate buffer. FIG. 178 shows the mAb C cumulative pool AR brokethrough the column of Capto Adhere operated at pH 8.5 and conductivityof 3.0 mS/cm with Tris-Acetate buffer. Both of graphs demonstratesimilar AR breakthrough curves with different AR values comparing toadalimumab (FIG. 176). FIG. 179 presents the AR breakthrough curves ofMab C with three different mixed mode resins with Tris-acetate bufferoperated at pH 8.5 and conductivity of 3.0 mS/cm. The data clearlydemonstrates that the AR reduction technology using mixed mode resinsworks very effectively for other antibodies.

Example MM 9.5 Demonstration of Relative pH on AR Reduction withDifferent Resins Using Adalimumab Antibody Material

In this Example, data compiled from different experiments is shown todemonstrate the impact of the pH choice, relative to the pI of theprotein on AR reduction. This data set provides the basis for oneskilled in the art to determine a pH range to implement the currentinvention. Further, this reiterates the fact that the pH choice dependson several factors and the relationship between pH and AR reduction isalso mAb dependent. FIG. 180 demonstrates the impact of pH-pI andconductivity on AR reduction which compiled data from the experimentsperformed with Capto Adhere under conditions listed in Table 60. FIG.181 shows the impact of pH-pI and conductivity on mAb B AR reductionincluding the experiments operated with Tris/Acetate buffer system andmultiple mixed mode resins under the conditions listed in Table 61. FIG.182 shows the impact of pH-pI and conductivity on mAb C AR reductionincluding the experiments operated with Tris/Acetate buffer system andmultiple mixed mode resins under the conditions listed in Table 62. Allthe load materials were from Protein A affinity capture and pH adjusted.It is also clear that the AR reduction can be achieved with the presentinvention with a range of pH choices, in the range of +0.5 to −2.5 pHunits from pI for adalimumab. One skilled in the art can choose anappropriate pH to achieve a target AR reduction.

TABLE 60 Operating conditions and AR reductions for adalimumab Buffersystem pH pH-pI Conductivity (mS/cm) AR reduction Tris/Ac 7 −2.02 4 0.47.6 −1.42 8 0.4 7.6 −1.42 2.3 1.3 7.6 −1.42 8 0.6 7.6 −1.42 8 0.2 7.6−1.42 8 −0.2 8.2 −0.82 4 2.1 7.6 −1.42 8 1.3 7 −2.02 12 −0.2 7.6 −1.42 81.2 8.2 −0.82 12 1.4 6.8 −2.27 8 1.2 8.4 −0.57 8 1.8 7.6 −1.42 8 1.4 7.6−1.42 13.7 1.0 7.6 −1.42 8 1.6 7.5 −1.52 3.75 1.7 7.6 −1.42 2.5 2.7 7.6−1.42 2.5 2.0 7.6 −1.42 5 1.3 7.6 −1.42 5 1.1 7.85 −1.17 2 3.5 7.85−1.17 3.75 3.2 7.85 −1.17 3.75 2.1 7.85 −1.17 3.75 2.8 7.85 −1.17 3.752.2 7.85 −1.17 5.5 2.1 8.1 −0.92 2.5 5.0 8.1 −0.92 2.5 2.6 8.1 −0.92 5−0.2 8.1 −0.92 5 −1.1 8.2 −0.82 3.75 2.9 Arg/Ac 8.5 −0.52 1 6.8 9.0−0.02 1 6.5 9.5 0.48 1 1.9 Trol/Ac 7.85 −1.17 1 5.7 8.0 −1.02 1 8.0 8.5−0.52 1 6.0

TABLE 61 Operating conditions and AR reductions for mAb B pH pH-pIConductivity (mS/cm) AR reduction Capto Adhere 6.8 −0.45 3 6.3 7 −0.25 36.2 7.5 0.25 3 4.0 8 0.75 3 3.2 6.8 −0.45 4.5 4.1 7.5 0.25 4.5 3.3 PPA6.8 −0.45 3 1.1 7 −0.25 3 0.9 7.5 0.25 3 1.3 8 0.75 3 0.5 6.8 −0.45 4.51.6 7.5 0.25 4.5 3.0 HEA 6.8 −0.45 3 1.8 7 −0.25 3 1.4 7.5 0.25 3 3.6 80.75 3 0.7 6.8 −0.45 4.5 2.2 7.5 0.25 4.5 0.9

TABLE 62 Operating conditions and AR reductions for mAb C pH pH-pIConductivity (mS/cm) Δ % AR Capto Adhere 8.0 −1.11 1 1.5 8.5 −0.61 1 3.59.0 −0.11 1 5.4 PPA 8.0 −1.11 1 −0.4 8.5 −0.61 1 1.1 9.0 −0.11 1 2.1 HEA8.0 −1.11 1 −1.6 8.5 −0.61 1 1.9 9.0 −0.11 1 2.8

Example MM 9.6 Effect of pH on AR Reduction

Response surface design DOE was applied to evaluate the impact of pH andconductivity on mAb AR reductions. In this example, adalimumab and CaptoAdhere were chosen. The experiments were performed with Tris/Acetatebuffer system. The load material was from Protein A affinity capture andpH adjusted. Besides the pH and conductivity ranged tested anddemonstrated in Table 63 and Table 64, higher pH ranges were alsostudied (FIG. 183).

The results in FIG. 183 and FIG. 184 demonstrated that mAb acidicspecies can be reduced at wide pH range from 6.8 to 9.5.

TABLE 63 DOE study condition Tris Acetate Edge points for ResponseBuffer Range Surface pH 7.0-8.2  6.8, 8.4 Conductivity 4.0-12.0 2.3,13.7

TABLE 64 AR reduction and Yield in DOE study Experiment # pHConductivity ΔAR Yield 1 7.0 4.0 0.4 83 2 7.6 8.0 0.4 73 3 7.6 2.3 1.382 4 7.6 8.0 0.6 68 5 7.6 8.0 0.2 70 6 7.6 8.0 −0.2 69 7 8.2 4.0 2.1 678 7.6 8.0 1.3 69 9 7.0 12.0 −0.2 70 10 7.6 8.0 1.2 71 11 8.2 12.0 1.4 7412 6.8 8.0 1.2 76 13 8.4 8.0 1.8 67 14 7.6 8.0 1.4 71 15 7.6 13.7 1.0 7416 7.6 8.0 1.6 70 Note: AR reductions and protein recovery yields werecalculated based on the Flow Through fractions at about loading 200 gprotein per L of resin

Example MM 9.7 Demonstration of AR Reduction with Different IonConcentrations/Ion Strength—Adalimumab

In this Example, adalimumab was chosen. Besides the conductivity rangetested presented before, lower conductivity and higher conductivityranges were also studied with the Capto Adhere. Table 65 and Table 66display the DOE study conditions using Capto Adhere columns withTris/Acetate buffer system. The load material was from Protein Aaffinity capture and pH adjusted. Column Flow Through pool was collectedin each run from 50 mAU of UV A280 on the ascending and 150 mAU on thedescending side of the peak. FIG. 185 demonstrates the effect of pH (6.8to 8.4), conductivity (2.3 to 13.7 mS/cm), and protein load amount (116to 354 g/L). FIG. 186 demonstrates the AR reduction at conductivity aslow as 1 mS/cm. Table 67 demonstrates the AR reduction at conductivity86 mS/cm with Ammonia Sulfate-Tris-Acetate buffer system.

The results demonstrated that mAb acidic species can be reduced at wideconductivity ranges from 1 to 86 MS/CM.

TABLE 65 DOE study condition Tris Acetate Edge points for ResponseBuffer Range Surface pH 7.6-8.1 7.5, 8.2 Conductivity 2.5-5.0 2.0, 5.5Protein load 150-320 116, 354 amount (g/L)

TABLE 66 DOE operating condition and results Conductivity Load amount pH(mS/cm) (g/L) ΔAR (%) Yield (%) 7.5 3.75 235 1.7 89 7.6 2.5 150 2.7 947.6 2.5 320 2.0 95 7.6 5 150 1.3 97 7.6 5 320 1.1 103 7.85 2 235 3.5 947.85 3.75 116 3.2 86 7.85 3.75 235 2.1 90 7.85 3.75 235 2.8 90 7.85 3.75354 2.2 91 7.85 5.5 235 2.1 92 8.1 2.5 150 5.0 80 8.1 2.5 320 2.6 87 8.15 150 −0.2 95 8.1 5 320 −1.1 98 8.2 3.75 235 2.9 90

TABLE 67 AR reduction and protein recovery at conductivity of 86 mS/cmand pH 7.9 Conductivity (mS/cm) pH Yield (%) ΔAR (%) 86 7.9 62 2.7 872.0 91 1.8 86 7.9 59 1.4 81 1.1 94 0.7

Note: adalimumab in Protein A eluate containing 25 mM acetate and 18 mMTris or 0.89 mM Tris were pH adjusted to pH 3.5 with 3M Acetic acidsolution and neutralized to pH 7.9 with 3M Tris solution. One part ofthis viral inactivated material was then diluted by adding 0.3 part of astock buffer containing 2.2 M (NH₄)₂SO₄/90 mM Tris/60 mM Acetic pH 7.9to reach conductivity of 86 mS/cm.

Example MM 9.8 Demonstration of AR Reduction with Different BufferSystems with Adalimumab

In this Example, adalimumab and resin Capto Adhere were chosen. Theexperiments were performed with different buffer systems listed in thetables below at multiple pH and conductivity condition. The loadmaterial pH was adjusted from Protein A eluate or CEX eluate. Theresults in Table 68 and Table 69 demonstrates that mAb acidic speciescan be reduced using various buffer systems.

TABLE 68 Effect of Cation type on mAb acidic species reduction andrecovery yield Tris/Acetae Operating Condition Capto Adhere HEA PPA pH7.85 pH 7.85 pH 7.85 4.5 mS/cm 3.5 mS/cm 2.5 mS/cm 4.5 mS/cm 3.5 mS/cm2.5 mS/cm 4.5 mS/cm 3.5 mS/cm 2.5 mS/cm % Yield 50 52 58 49 52 56 40 6463 Δ % 1.8 3.8 3.7 1.1 2.7 3.2 1.4 1.9 2.6 AR Arginine/Acetate OperatingCondition ~1 mS/cm ~1 mS/cm ~1 mS/cm pH 8.5 pH 9.0 pH 9.5 pH 8.5 pH 9.0pH 9.5 pH 8.5 pH 9.0 pH 9.5 % Yield 65 62 49 77 71 66 69 70 71 Δ % 8.66.5 1.9 4.9 3.5 N/R 4.5 1.9 0.6 AR Trolamine/Acetate Operating Condition~1 mS/cm ~1 mS/cm ~1 mS/cm pH 7.85 pH 8.0 pH 8.5 pH 7.85 pH 8.0 pH 8.5pH 7.85 pH 8.0 pH 8.5 % Yield 62 54 49 69 64 58 64 64 590 Δ % 4.1 6.04.6 1.7 2.9 3.0 1.4 2.1 2.1 AR Note: Load material was adalimumab fromProtein A affinity capture and pH adjusted

TABLE 69 Effect of Anion type on mAb acidic species reduction andrecovery yield load amt conductivity Yield Δ % Buffer (g/L) (mS/cm) pH(%) AR Tris/Acetate¹ 200 4.00 7.80 90 1.6 NaPhosphaste/Citrate/ 200 3.537.87 87 1.5 Trolamine/NaCl² Tris/Formate¹ 300 0.92 8.50 69 3.7 ¹Loadmaterial was adalimumab from Protein A affinity capture and pH adjusted²The load material was adalimumab from CEX capture and pH adjusted

Example MM 9.9 Demonstration of AR Reduction with Different Loading

The experiments were performed with Tris/Acetate buffer system under theconditions in Table 66. The load material was adalimumab from Protein Aaffinity capture and pH adjusted. Column Flow Through pool was collectedin each run from 50 mAU of UV A280 on the ascending and 150 mAU on thedescending side of the peak. As seen from the profile (FIG. 186), theloading capacity has an impact on AR reduction but the AR reduction canbe achieved over a wide range of loading capacities, and is merely atrade-off between AR reduction and recovery.

Example MM 9.10 Demonstration of AR Reduction with Different LoadConcentration

In this example, Capto Adhere was chosen. The experiment was performedwith Tris/Acetate buffer system at pH 7.8±0.1 and conductivity 3.0±0.05mS/cm. The load material was adalimumab from concentrated CEX captureand pH adjusted. The prepared load material was then split to be twoparts. One was directly loaded on to a Capto adhere column; the otherpart was diluted 2 folds with equilibration buffer to make differentprotein concentration. Table 70 demonstrates that the load proteinconcentration did not have significant impact on mAb acidic speciesreduction.

TABLE 70 Adalimumab AR Reduction and Yield with Different Load ProteinConcentration Load Load protein Capture amount Conductivity conc. YieldΔ % step Buffer (g/L) (mS/cm) pH (g/L) (%) AR CEX Tris/Acetate 200 2.97.8 22.0 87 2.4 CEX Tris/Acetate 200 3.0 7.7 11.0 89 2.1 CEXNaPhiosphaste/ 200 3.5 7.9 4.9 87 1.5 Citrate/Trolamine/ NaCl Protein ATris/Acetate 200 3.1 7.8 9.0 89 2.5 Protein A Tris/Acetate 200 4.0 7.811.8 90 1.6 Protein A Tris/Acetate 200 3.0 7.8 9.9 93 2.4 Protein ATris/Acetate 208 3.0 7.8 8.4 95 3.2 Protein A Tris/Acetate 222 3.0 7.912.9 89 3.4

Example MM 9.11 Alternative Wash Modalities

In this example, mAb adalimumab and resin Capto Adhere were chosen. Theexperiments were performed with Tris/acetate buffer system and the loadmaterial pH was adjusted from Protein A eluates. The equilibrationbuffer for both run was Tris/Acetic acid pH 7.8±0.1 and conductivity of3.0±0.1 mS/cm. In the gradient conductivity wash study, second bufferwas Tris/Acetic acid pH 7.8±0.1 and conductivity 6.0 mS/cm.

The results demonstrated that post load pH and conductivity can bevaried with minimal AR reduction impacted (see Table 71).

TABLE 71 Comparison of AR reduction and yield under different washconditions Load Load conductivity load conc Yield Wash Δ % ExperimentWash (mS/cm) pH (mg/mL) (%) CV AR Equilibration Equilibration buffer3.09 7.85 9.04 89 16.4 2.5 buffer wash (Tris/Ac pH 7.8 and 3.0 mS/cm)wash only Gradient conductivity 1CV Equilibration 3.04 7.78 7.17 91 8.02.2 wash buffer 10CV gradient conductivity wash from 100% Tris/Ac pH7.8, 3.0 mS/cm to 100% Tris/Ac pH 7.8, 6 mS/cm,

Example MM 9.12 Demonstration of Achievement of Absolute Value of ARLevels in Antibody Preparations Using Mixed Mode Chromatography

In this example, mAb adalimumab was chosen. The experiments wereperformed with multiple buffer systems and multiple MM absorbents underconditions listed in Table 72. The load materials pH was adjusted fromProtein A eluates.

As described above, the AR for adalimumab is further grouped into tworegions termed AR1 and AR2, based on a certain retention time of thepeaks seen on the WCX-10 method. The characteristics of the variants inthese two regions are expected to be different and hence the methodsthat reduce variants belonging to these groups can be specificallydelineated. Further, in addition to achieving a certain AR reduction, itmay be desirable to achieve a certain absolute level of AR levels, inconsideration of reducing or removing certain variants. The capabilityof the current invention in achieving a certain absolute level of AR,AR1 and AR2 is demonstrated in Table 72.

TABLE 72 Acidic species level in MM resin Flow Through Resin Buffer pHConductivity (mS/cm) Yield (%) FT % AR1 FT % AR2 Capto AdhereTris/Acetate 7.85 4.5 50 2.8 9.7 7.85 4.5 68 3.0 10.3 7.85 3.5 52 1.610.0 7.85 3.5 71 2.2 10.5 7.85 3.0 93 3.2 9.7 7.85 2.5 58 1.7 9.4 7.852.5 72 2.2 10.0 Arginine/Acetate 8.5 1 65 1.2 6.1 9.0 1 62 1.6 7.2 9.5 149 0.8 11.8 Trolamine/Acetate 7.9 1 44 1.5 6.6 7.9 1 62 1.8 8.0 8.0 1 371.1 5.8 8.0 1 54 1.2 7.7 8.5 1 32 1.7 9.0 8.5 1 49 1.9 10.1 Tris/Formate8.5 1 69 0.6 6.4 HEA Arginine/Acetate 8.5 1 77 1.6 8.5 9.0 1 71 0.8 12.0PPA Arginine/Acetate 8.5 1 69 2.2 8.7 9.0 1 70 1.0 13.5 9.5 1 71 0.713.1

Example MM 9.13 Demonstration of HCP and Aggregate Reduction in Additionto AR Reduction

Besides the acidic species reduction, the MM adsorbent is able to reduceother product/process related substances/impurities effectively. In theimplementation of the current invention the fact that AR reduction iseffected, other impurities/substances are expected to be clearedsignificantly as they should bind stronger than the acidic species. Thedata shown in Table 73 and Table 74 demonstrates significant HCP andaggregate reductions with different resins, buffer systems, pH,conductivities and molecules

TABLE 73 Aggregate reduction Conduc- tivity (mS/cm) pH Buffer medium Δ %HMW adalimumab 3.75 7.5 Tris/Acetate Capto Adhere 0.7 2.5 7.6 0.9 2 7.850.9 3.75 7.85 1.0 5.5 7.85 0.7 2.5 8.1 1.0 3.75 8.2 0.8 4.0 8.2 1.0 8.06.8 0.2 8.0 8.4 1.0 1.0 8.5 Arginine/Acetate Capto Adhere 0.5 1.0 9.00.8 1.0 9.5 0.9 1.0 8.5 HEA 0.4 1.0 9.0 2.5 1.0 9.5 0.7 1.0 8.5 PPA 0.51.0 9.0 2.8 1.0 9.5 0.4 mAb C 3.0 8 Tris/Acetate Capto Adhere 1.0 3.08.5 Capto Adhere 1.1 3.0 9 Capto Adhere 0.6 3.0 8 PPA 0.7 3.0 8.5 PPA0.5 3.0 8 HEA 0.7 3.0 8.5 HEA 0.6

TABLE 74 HCP Log reduction Con- ductivity (mS/cm) pH Buffer medium HCPLRF adalimumab 3.75 7.5 Tris/Acetate Capto Adhere 1.5 2.5 7.6 1.7 2.07.85 2.2 3.75 7.85 1.9 5.5 7.85 1.4 2.5 8.1 2.3 3.75 8.2 2.1 4.0 8.2 1.78.0 6.8 0.3 8.0 8.4 0.7 mAb B 3 6.8 Capto Adhere 2.0 4.5 6.8 1.3 3 6.8PPA 1.2 4.5 6.8 1.2 3 6.8 HEA 1.3 4.5 6.8 1.1

Example MM 9.14 Combinations of MM with Alternative SeparationStrategies

Acidic species reduction by MM adsorbents is expected to be performedafter capture of the antibody by other means, or after one or moreintermediate steps following the capture step. In the Examples below theMM adsorbent steps were performed either following a Cation ExchangeCapture step or Protein A affinity capture step. As shown in Table 75,AR reduction was achieved at two different conductivities followingProtein A Chromatography and CEX Chromatography.

TABLE 75 AR Reduction with different source materials Yield Δ % CaptureBuffer conductivity (mS/cm) pH (%) AR Protein A Tris/Acetate 3.1 7.8 892.5 Protein A 4.0 7.8 90 1.6 CEX 2.9 7.8 87 2.4 CEX 3.0 7.7 89 2.1

Adalimumab was purified by a CEX chromatography step followed with a lowpH viral inactivation step. The filtered viral inactivated material wasbuffer exchanged and loaded onto a Capto Adhere column. The Flow Throughof Capto Adhere material was then purified with a HIC column withbind/elute mode. As shown in Table 76, AR reduction was achievedprimarily with MM step, with some contribution from other steps.

TABLE 76 Complete Process train with CEX Chromatography Capture- ARReduction Δ % AR Δ % Lys Yield (%) CEX eluate n/a n/a n/a MM Load 0.290.34 90% MM Flow Through 2.57 2.57 93% HIC eluate 0.95 0.94 97%

Adalimumab was purified using a Protein A chromatography step followedwith a low pH viral inactivation step. The filtered viral inactivatedmaterial was buffer exchanged and loaded onto a Capto Adhere column. TheFlow Through of Capto Adhere material was then purified with a HICcolumn with bind/elute mode as well as Flow Through mode. As shown inTable 77, AR reduction was achieved primarily with MM step, with somecontribution from other steps.

TABLE 77 Complete Process Train with Protein A Capture - AR, HMW and HCPreduction % AR % HMW Process Yield (%) reduction reduction HCP LRFClarified Harvest 97.0% n/a n/a n/a Prt-A Eluate Pool 89.6% 0.06 1.87Viral Inactivated 99.7% No reduction 0.07 0.39 Filtrate MM FT pool 91.9%2.26 0.83 1.63 HIC (B/E) Eluate 90.1% 0.40 0.22 1.41 Nanofiltrate 90.7%No reduction No reduction 0.15 Filtrate BDS (B/E) 102.0% No reduction Noreduction 0.22 HIC FT-pool 98.5% 0.16 0.23 0.46 VF(FT) Filtrate 96.1% Noreduction No reduction 0.10 BDS (FT) 103.8% No reduction No reduction Noreduction

Example 10 Upstream and Downstream Process Combinations to AchieveTarget % AR or AR Reductions

The instant example demonstrates the combined effect of one or moreupstream and downstream process technology in achieving a target ARvalue or AR reduction, thereby facilitating the preparation of anantibody composition having a specific charge heterogeneity.

Example 10.1 Combination of Upstream and Downstream Technologies UsingMM

In this Example, the combination of upstream and downstream methodsinvolves the reduction of acidic species in 3 L bioreactor cell culturessupplemented with arginine (2 g/L) and lysine (4 g/L). The results ofthat strategy are summarized in Table 78. The total acidic species wasreduced from 20.5% in the control sample to 10.2% in sample fromcultures that were supplemented with the additives.

In this study, adalimumab producing cell line 1 was cultured in media 1(chemically defined media) supplemented with amino acid arginine (2 g/L)and lysine (4 g/L) in a 300 L bioreactor. On Day 12 of culture, theculture was harvested and then subsequently analyzed using WCX-10 postProtein A purification and the percentages of total peak(s) areacorresponding to the acidic species were quantified. The percentage ofacidic species was estimated to be 9.1% in the 300 L harvest sample.

TABLE 78 AR levels achieved with use of upstream technologies 3 LBioreactor 300 L Bioreactor Arginine (2 g/L) + Arginine (2 g/L) +Control Lysine (4 g/L) Lysine (4 g/L) AR1 AR2 Total AR1 AR2 Total AR1AR2 Total (%) (%) AR (%) (%) (%) AR (%) (%) (%) AR (%) 6.3 14.2 20.5 2.67.6 10.2 2.4 6.7 9.1

The material produced by the 300 L Bioreactor employing arginine andlysine additions, that effectively reduced the AR levels to 9.1% waspurified using a downstream process employing Mixed Mode chromatographyas the primary AR reduction method.

Adalimumab was purified by a Protein A chromatography step followed witha low pH viral inactivation step. The filtered viral inactivatedmaterial was buffer exchanged and loaded onto a Capto Adhere column. TheFlow Through of Capto Adhere material was then purified with a HICcolumn with bind/elute mode as well as Flow Through mode. As shown inTable 79, AR reduction was achieved primarily with MM step, with somecontribution from other steps. The table also shows that additionalproduct related substances such as aggregates and process relatedimpurities such as HCP can be effectively reduced employing thesecombined technologies.

TABLE 79 Complete Downstream Process Train with Protein A Capture - AR,HMW and HCP reduction % AR % HMW Process Yield (%) reduction reductionHCP LRF Clarified Harvest 97.0% n/a n/a n/a Prt-A Eluate Pool 89.6% 0.061.87 Viral Inactivated 99.7% No reduction 0.07 0.39 Filtrate MM FT pool91.9% 2.26 0.83 1.63 HIC (B/E) Eluate 90.1% 0.40 0.22 1.41 Nanofiltrate90.7% No reduction No reduction 0.15 Filtrate BDS (B/E) 102.0% Noreduction No reduction 0.22 HIC FT-pool 98.5% 0.16 0.23 0.46 VF(FT)Filtrate 96.1% No reduction No reduction 0.10 BDS (FT) 103.8% Noreduction No reduction No reduction

As is evident from the above example, the MM method further reduced theAR levels by 2.26%. Therefore upstream technologies for reduction can becombined with downstream technologies to achieve desired AR levels/ARreduction.

Example 10.2 Demonstration of AR Reduction in Process Combinations

The methods described above for reducing acidic species using cationexchange can be used as an independent operation or in combination withother process steps that provide additional acidic species reduction orthose providing additional complementary and supplementary purification(See Tables 80-87). The following process combinations are provided hereas non-limiting examples

1. Affinity→MM→CEX

2. Affinity→AEX→CEX

3. Affinity→CEX

4. CEX Capture→CEX

TABLE 80 AR Reduction by Capto Adhere (mixed mode) followed by Poros XS(CEX) % % AR1 % AR Step Yield % AR1 % AR Reduction Reduction MabSureEluate 2.90 10.08 Viral Inact 89 2.89 10.42 Mixed Mode FTW 94 2.26 8.520.64 1.90 CEX Load 2.29 8.97 CEX Eluate 91 0.25 4.88 2.04 4.10 Overall76 2.65 5.20

TABLE 81 Aggregate reduction by combination of Capto Adhere (mix mode)Poros XS (CEX) % % % % Mono % Agg. % Frag Step Yield % Monomer AggregateFragment increase decrease decrease MabSure 99.08 0.85 0.08 Eluate ViralInact 89 99.14 0.73 0.13 Mixed Mode 96 99.64 0.26 0.10 0.50 0.47 0.03FTW CEX Load 99.64 0.26 0.10 CEX Eluate 89 99.74 0.18 0.08 0.10 0.080.02 overall 76 0.66 0.67 0.00

TABLE 82 AR Reduction by Poros PI (AEX) followed by Poros XS (CEX) AEXCEX Cycle C % % AR1 % AR Step Yield % AR1 % AR Reduction ReductionMabSure Eluate 2.90 10.08 AEX Load 2.73 10.16 AEX FTW 90 1.64 6.7 1.093.46 Viral Inact 100 1.39 6.03 CEX Load 2.76 6.18 CEX Eluate 91 0.153.22 2.61 2.96 Overall 82 2.75 6.86

TABLE 83 Aggregate reduction Poros PI (AEX) Poros XS (CEX) % AEX CEXCycle C % % % Mono % Agg. % Frag Step Yield % Monomer Aggregate Fragmentincrease decrease decrease MabSure 99.08 0.85 0.08 Eluate AEX Load 98.671.25 0.03 AEX FTW 90 99.88 0.05 0.07 1.21 1.2 −0.04 Viral Inact 10099.94 0.05 0.02 CEX Load 99.64 0.26 0.10 CEX Eluate 91 99.79 0.13 0.080.14 0.13 0.02 Overall 82 0.71 0.72 0.00

TABLE 84 AR reduction from an Affinity capture pool followed by Poros XS(CEX) % % AR1 % AR Step Yield % AR1 % AR Reduction Reduction MabSureEluate 3.0 10.5 CEX Eluate 82.7 0.3 4.9 2.8 5.6

TABLE 85 Aggregate reduction: Affinity capture pool followed by Poros XS(CEX) % % % % Mono % Agg. % Frag Step Yield % Monomer Aggregate Fragmentincrease decrease decrease MabSure 98.5 1.4 0.1 Eluate CEX Eluate 82.799.7 0.2 0.1 1.2 1.2 0.0

TABLE 86 AR reduction CEX Capture (Fractogel SO3) followed by Poros XS(CEX) 145 mM TA Poros XS adalimumab % % AR1 % AR Step Yield % AR1 % ARReduction Reduction Concentrated Fractogel 3.3 14.0 Eluate VI CEX Eluate72.6 0.44 6.7 2.8 7.3

TABLE 87 Aggregate reduction: CEX Capture (Fractogel) followed by PorosXS (CEX) 145 mM TA Poros XS adalimumab % % % % Mono % Agg. % Frag StepYield % Monomer Aggregate Fragment increase decrease decreaseConcentrated 97.9 1.5 0.7 Fractogel Eluate VI CEX Eluate 72.6 98.7 1.10.2 0.9 0.4 0.5

Example 10.3 Process Combination: Protein A, AEX, CEX Combination withTris/Formate Buffer System

In Example 10.3, AR reduction through a process combination of Protein Aaffinity capture followed by fine purification with AEX and CEXchromatography in a Tris/Formate buffer system was examined.

Materials and Methods

Materials

Antibody

Adalimumab monoclonal antibody preparation was obtained after affinitycapture of the clarified harvest. The eluate from the capture step wasbuffer exchanged as required.

AEX Adsorbent:

Poros 50HQ (Applied Biosciences, part#1-2459-11), a rigid 50 μmpolymeric bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene] was used in this experiment.

CEX Adsorbent:

Poros XS (Applied Biosciences, part#4404338), a rigid 50 μm polymericbead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene], was used in this experiment.

Methods

AEX Chromatography Method

Poros 50HQ was packed in 1.0 cm×10.0 cm (OmniFit) columns. The columnwas equilibrated in a buffer system with appropriate pH andconductivity. The load was prepared in the equilibration buffer byaddition of the stock solutions to obtain the target ion concentrations,as specified, and loaded on the column, as specified, followed bywashing with the equilibration buffer for 20 CV. The antibody productwas collected in the flow-through and wash fractions during the load andwashing steps. The columns/housings were then regenerated with 100 mMformate and 1M of NaOH solution was used for column cleaning.

CEX Chromatography Method

Poros XS was packed in 1.0 cm×10.0 cm (OmniFit) columns. The column wasequilibrated in a buffer system with appropriate pH and conductivity.The column load was prepared in the equilibration buffer by bufferexchange or addition of the stock solutions to obtain the target ionconcentrations as specified and loaded on the column at approximately 40g protein/L resin (or as specified) followed by washing with theequilibration buffer for 20 CV (or as specified). The antibody productwas then eluted, and the column regenerated.

Buffer Preparation Method

Buffers for AEX were prepared targeting a specific ion concentration forthe anion by fixing the anion concentration (acid) to the target value,and adjusting the solution with the cationic component (base) to achievethe appropriate pH. For example, to prepare a 10 mM Formate-Tris buffersolution, pH 8.7, formic acid was dissolved in water to a targetconcentration of 10 mM and adjusted with concentrated Tris-base to pH8.7.

Buffers for CEX were prepared targeting a specific ion concentration forthe cation by fixing the cation concentration (base) to the targetvalue, and adjusting the solution with the anionic component (base) toachieve the appropriate pH. For example to prepare a 140 mM Tris-Formatebuffer solution, pH 7.5, Tris base was dissolved in water to a targetconcentration of 140 mM and adjusted with Formic Acid to pH 7.5.

AR Reduction and Recovery Calculations

In general, eluate fractions and Flow Through (FT)/Wash fractions werecollected and analyzed with a WCX-10 method for AR levels. By actual orcalculated pooling of the fractions the recovery and the correspondingAR levels were calculated.

Analytical Methods

WCX-10 for Adalimumab

The acidic species and other charge variants present in the adalimumabprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 10 mM SodiumPhosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphatedibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binarygradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B:22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.

Quantitation was based on the relative area percent of detected peaks.The peaks that elute at relative residence time less than a certain timeare together represented as the acidic peaks.

Size Exclusion Chromatography

The molecular-weight distribution of collected samples was quantifiedaccording to the following methods. Size exclusion chromatography (SEC)was performed using a TSK-gel G3000SW×L, 5 μm, 125 Å, 7.8×300 mm column(Tosoh Bioscience) on an HP Agilent HPLC system. Injections were madeunder isocratic elution conditions using a mobile phase of 200 mM sodiumsulfate, 100 mM sodium phosphate, pH 6.8, and detected with absorbanceat 214 nm. Quantification was based on the relative area of detectedpeaks.

UV spectroscopy A₂₈₀

UV A280 was used to determine protein concentrations for the samplespost protein A elution. The assay was performed on an Agilent UVSpectrophotometer. The protein concentration was determined usingBeer-Lambert's Law, A=dc, where A is Absorbance, c is the extinctioncoefficient, 1 is the path length, and c is the concentration. Theabsorbance was taken at 280 nm, the path length was 1 cm, and theextinction coefficients were 1.39 for Adalimumab, 1.38 for mAb B, and1.43 for mAb C.

Results

AR reduction through a process combination of Protein A affinity capturefollowed by fine purification with Poros 50HQ and Poros XS in aTris/Formate buffer system was carried out as follows, resulting in afinal AR of 1.4%. This exemplary low AR process followed the flow pathset forth in FIG. 190.

Protein A

For Protein A affinity capture, a 2.2×20 cm MabSelect SuRe (GEHealthcare) column was packed and qualified by HETP/Asymmetry analysis.The chromatography was run in bind-elute mode with a 4-minute residencetime. Columns were loaded with 37 g mAb protein per liter of resin.

The column was washed with a high concentration Tris/Formate buffer,rinsed with a low concentration Tris/Formate buffer and subsequentlyeluted with a low pH Tris/Formate buffer. The column was thenregenerated and cleaned with hydroxide solutions appropriate for theresin.

The MabSelect SuRe™ eluate pool was titrated to pH 3.7 with formic acidand held for an hour. The acidified materials were mixed for 1 hour atambient temperature. The VI pool was neutralized with to a pH of 8.7(i.e., AEX Load). The AEX load was filtered prior to loading.

AEX Chromatography

All AEX chromatography experiments were carried out on an AKTAavant25system using a 1.0 cm diameter×9.5 cm length column packed with Poros50HQ resin, and qualified by HETP/Asymmetry analysis. Each experimentwas performed at ambient temperature. The AEX step was performed at 225g/L of resin loading. Equilibration and loading was performed with a lowconcentration Formate/Tris buffer, e.g., a 15 mM Formate/Tris buffer ata pH of 8.7. Wash was performed with Acetate and Tris at the same pH.Each run was performed at ambient temperature with a load concentrationof ˜10 g/L at a residence time of 3 minutes. The column was regeneratedand cleaned with solutions appropriate for the resin.

The Flow Through was collected in the following fractions: 100 mAu-175g/L, 175 g/L-200 g/L, 200 g/L-225 g/L+1 CV of wash. The fractions werethen measured by A280 mass spectroscopy and analyzed by WCX-10 and SECassays.

Poros 50HQ FTW pool was adjusted to 135 mM Tris/Formate pH 7.5 usingstock solutions of Tris and Formic acid.

CEX Chromatography

All CEX chromatography experiments were carried out on an AKTAavant150system using a 1.0 cm diameter×11 cm length column packed with Poros XSresin, and qualified by HETP/Asymmetry analysis. Each experiment wasperformed at ambient temperature, with a 5.8 mg/mL load, 40 g\L resinloading, and a residence time of 6 minutes. Equilibration, loading, andwash was performed with a high concentration Tris/Formate buffer at a pHof 7.5. Elution was with sodium sulfate and Tris/Formate buffers. Theeluate was collected in one fraction from 400 mAU to 100 mAU. Threecycles were performed. The column was regenerated and cleaned withsolutions appropriate for the resin.

Viral filtration was performed on the Poros XS Eluate before the UFDFprocessing, using a Virosart CPV Viral Filter.

Ultracel 3 Biomax 30-kDa filters were used for diafiltration (intowater) and concentration of the CEX Eluate.

The cumulative AR of the Poros50HQ fractions was below 6% allowing themto be pooled together, and adjusted to CEX Load conditions. Three cyclesof CEX were performed. All three CEX eluate cycles had an AR below 3%and a HMW below 0.2% and were pooled together.

The step yield for each unit operation is listed in Table 88 with afinal overall yield of 38% being achieved. The process was able toachieve an adalimumab composition with a final AR of 1.4% (an AR1 of0.0% and an AR2 of 1.4%) and final HMW of 0.10%.

TABLE 88 Step Yields for Low AR Material Generation Step Yield AR % AR1% AR2 % HMW MabSuRe 86% 10.0% 1.6% 8.4% NA Poros 50HQ-FTW 80% 5.4% 0.8%4.6% NA Poros 50XS 55% 1.4% 0.0% 1.4% 0.13% Overall 38% 1.4% 0.0% 1.4%0.10%

Example 11 AR Reduction Using “Recycled” AEX and CEX Technologies

This Example describes the “recycle” mode of chromatography for ARreduction using AEX, CEX, and MM technologies.

Materials and Methods

Materials

Antibody

Adalimumab monoclonal antibody preparation was material obtained afteraffinity capture of a clarified harvest. The eluate from the capturestep was buffer exchanged as required.

AEX Adsorbent:

Poros 50HQ (Applied Biosciences, part#1-2459-11), a rigid 50 μmpolymeric bead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene], was used in this experiment.

CEX Adsorbent:

Poros XS (Applied Biosciences, part#4404338), a rigid 50 μm polymericbead with a backbone consisting of cross-linkedpoly[styrene-divinylbenzene], was used in this experiment.

Methods

AEX Chromatography Method

Poros 50HQ was packed in 1.0 cm×10.0 cm (OmniFit) columns. The columnwas equilibrated in a buffer system with appropriate pH andconductivity. The load was prepared in the equilibration buffer byaddition of the stock solutions to obtain the target ion concentrationsand loaded on the column, followed by washing with the equilibrationbuffer for 20 CV. The antibody product was collected in the flow-throughand wash fractions during the load and washing steps. Thecolumns/housings were then regenerated with 100 mM formate and 1M ofNaOH solution was used for column cleaning.

CEX Chromatography Method

Poros XS was packed in 1.0 cm×10.0 cm (OmniFit) columns. The column wasequilibrated in a buffer system with appropriate pH and conductivity.The column load was prepared in the equilibration buffer by bufferexchange or addition of the stock solutions to obtain the target ionconcentrations as specified and loaded on the column at approximately 40g protein/L resin (or as specified) followed by washing with theequilibration buffer for 20 CV (or as specified). The antibody productwas then eluted, and the column regenerated.

Buffer Preparation Method

Buffers for AEX were prepared targeting specific ion concentration forthe anion by fixing the anion concentration (acid) to the target value,and adjusting the solution with the cationic component (base) to achievethe appropriate pH. For example, to prepare a 10 mM Formate-Tris buffersolution, pH 8.7, formic acid was dissolved in water to a targetconcentration of 10 mM and adjusted with concentrated Tris-base to pH8.7.

Buffers for CEX were prepared targeting specific ion concentration forthe cation by fixing the cation concentration (base) to the targetvalue, and adjusting the solution with the anionic component (base) toachieve the appropriate pH. For example to prepare a 140 mM Tris-Formatebuffer solution, pH 7.5, Tris base was dissolved in water to a targetconcentration of 140 mM and adjusted with Formic Acid to pH 7.5.

AR Reduction and Recovery Calculations

In general, eluate fractions and Flow Through/wash fractions werecollected and analyzed with WCX-10 method for AR levels. By actual orcalculated pooling of the fractions the recovery and the correspondingAR levels were calculated.

Analytical Methods

WCX-10 for Adalimumab

The acidic species and other charge variants present in the adalimumabprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 10 mM SodiumPhosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphatedibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binarygradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B:22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.

Quantitation was based on the relative area percent of detected peaks.The peaks that elute at relative residence time less than a certain timeare represented together as the acidic peaks.

Size Exclusion Chromatography

The molecular-weight distribution of collected samples was quantifiedaccording to the following methods. Size exclusion chromatography (SEC)was performed using a TSK-gel G3000SW×L, 5 μm, 125 Å, 7.8×300 mm column(Tosoh Bioscience) on an HP Agilent HPLC system. Injections were madeunder isocratic elution conditions using a mobile phase of 200 mM sodiumsulfate, 100 mM sodium phosphate, pH 6.8, and detected with absorbanceat 214 nm. Quantification is based on the relative area of detectedpeaks.

UV spectroscopy A₂₈₀

UV A280 spectroscopy was used to determine protein concentrations forthe samples post Protein A elution. The assay was performed on anAgilent UV Spectrophotometer. The protein concentration was determinedusing Beer-Lambert's Law, A=dc, where A is Absorbance, ε is theextinction coefficient, 1 is the path length, and c is theconcentration. The absorbance was taken at 280 nm, the path length was 1cm, and the extinction coefficients were 1.39 for adalimumab, 1.38 formAb B, and 1.43 for mAb C.

Example 11.1 Recycled AEX Chromatography

In this Example, a cycling strategy was employed to increase therecovery yield for a given target product quality attribute. The ARreduction for a given Formate concentration and pH can be modulated byadjusting the load. Also, the recovery yield is fixed for a givenloading. In this strategy, the load is chosen to achieve a target ARlevel in the Flow Through fraction. The column is then eluted with aFormate concentration that is slightly higher than the load. The elutateis collected, and then diluted with water to match the load Formateconcentration, and added back into the load tank. This column cycle isrepeated several times (and is referred to as “recycled”chromatography).

For this experiment, the target AR level in the Flow Through (FT) poolwas set at 5%. The Poros50HQ column was first loaded to 200 g/L of resinand the Flow Through was collected at 20 g/L of protein loaded on theresin with the equilibration/wash buffer and load condition 15 mMAcetate/Tris pH8.7. The Flow Through fractions were run on WCX-10 assayand the cumulative AR breakthrough was calculated. The cumulative ARbreakthrough of 5% was observed to occur at 150 g/L of protein loadedonto the resin and all the subsequent experiments were run at 150 g/Lloading.

The cycling phase involved the scheme detailed in Table 89. The AEX loadwas prepared by adjusting the MabSelect SuRe eluate with 3M Tris to theappropriate pH and diluted to 15 mM Acetate and then filtered. The FlowThrough of the load and wash were collected in two separate vessels. Thewash was spiked with enough MabSelect SuRe Eluate to perform anothercycle at 150 g/L and the condition was adjusted to 15 mM Acetate/TrispH8.7. A total of 4 cycles were performed using the sequence of stepsdescribed above. Each run was performed at ambient temperature with aresidence time of 3 minutes following the chromatographic conditionslisted in Table 89. The flow-through was collected in one fraction from100 mAu until the end of step, and the wash was collected from thebeginning of the step to 50 mAU. The FT Wash was then measured by A280and analyzed by the WCX-10, and SEC assays.

TABLE 89 AEX Chromatography Conditions Column Step Solution VolumesEquilibration* 15 mM Acetate/Tris pH 8.7 30 Load* adalimumab ~15 mMAcetate/Tris 150 g/L of resin pH 8.7 Wash* 30 mM Acetate/Tris pH 8.5Wash down to 50 mAu Regeneration 100 mM Acetate/Tris + 500 mM  5 NaCl pH3.5

Cycling the wash fraction on the AEX column as a means of controllingthe level of process impurities was implemented in this study. The washfraction was collected at each cycle(C_(n)) and adjusted to properloading conditions and loaded at the subsequent cycle (C_(n+1)). Theloading amount was dialed in to provide an AR breakthrough of 5%. Atotal of four cycles were performed.

TABLE 90 AEX Cycling Product Quality Load Wash Load Wash Yield AR FT ARAR Regen AR Lys FT Lys Lys Regen Lys Cycle (%) (%) (%) (%) (%) (%) (%)(%) (%) 1 64.8 13.2 5.1 24.8 89.1 85.8 93.7 74.3 10.3 2 65.3 16.5 6.133.9 90.1 82.6 92.9 65.2 9.0 3 58.5 18.6 5.9 36.1 90.1 80.6 93.0 63.09.1 4 58.2 18.4 5.9 38.4 85.1 80.8 93.1 60.8 8.8

The step yield and product quality is listed in Table 90. The % lysine(sum lysine variants, i.e., Lys 0, Lys 1 and Lys 2 which are mAbscontaining 0, 1 or 2 terminal lysines) is the quantitation of thedesired (non-AR containing) fraction of the product and is provided hereto show that the recycle method is able to recover over 93% of thedesired product. Product containing higher levels of AR is recovered inthe Wash fraction of each cycle, which is then recycled back onto thesubsequent AEX cycle. The recycling of the wash fraction improves thecumulative yield and while maintaining the product quality as shown inTable 91. The Cumulative Yield increased from 65% to 81% in the fourcycles, while maintaining the AR level at ˜6% and the monomer level at˜99.4%.

TABLE 91 Cumulative Product Quality for AEX Cycling CumulativeCumulative Cumulative Cumulative Cumulative Cumulative Monomer CycleYield (%) AR (%) AR1 (%) AR2 (%) Lys (%) (%) 1 65 5.1 0.6 4.5 93.7 99.42 77 5.6 0.6 5.6 93.3 99.5 3 79 5.7 0.7 5.1 93.2 99.4 4 81 5.8 0.7 5.193.2 99.3

Example 11.2 Recycled CEX Chromatography

These experiments were performed using Protein A eluate as CEX loadingmaterial. Cycle 1 (control) was performed under load/wash bufferconditions of 160 mM tris-acetatet, pH 7.5, 40 g protein/L resin. Cycle2 was performed by combining part of the wash from Cycle 1 and freshProtein A eluate as loading material. The earlier wash (prior toreaching the peak) which contained higher AR was discarded and the restof the wash was included in the load. The loading and wash conditionswere the same as Cycle 1. Cycle 3 and Cycle 4 were performed the sameway as Cycle 2.

The results shown in Table 92 indicate that the recycle chromatographywith four runs increase the yield from 53.4% to 65.1%. AR reduction forCycle 1 is 8.84% and whereas with the 4 cycle Recycle Chromatography is7.79%. While achieving similar product quality, the recyclechromatography approach can significantly improve the yield.

TABLE 92 Recycle Chromatography impact on AR reduction and yield RecycleProduct Quality Cycle 1 Cycle 2 Cycle 3 Cycle 4 Chrom. Yield (%) 53.452.2 52.1 51.6 65.1 % AR1 in load 3.75 3.71 3.31 3.40 n/a % AR1 ineluate 0.02 0.08 0.05 0.02 0.03 % AR 1 reduction 3.72 3.64 3.26 3.383.72 % AR2 in load 9.6 11.5 12.0 12.0 n/a % AR2 in eluate 4.48 5.61 6.006.06 5.52 % AR 2 reduction 5.11 5.88 5.96 5.98 4.07 Total AR (%) in 13.315.2 15.3 15.4 n/a load Total AR (%) in 4.51 5.69 6.05 6.08 5.55 eluate% Total AR 8.84 9.51 9.22 9.36 7.79 reduction

Example 11.3 AR Reduction Using MM Recycled Chromatography

The following Materials and Methods were used for Example 11.3.

Materials and Methods

Material captured by Protein A affinity chromatography

Adalimumab clarified harvest material obtained from 300 L bioreactor(SUL101912) was loaded on a Protein A affinity column chromatography(such as MabSelect SuRe) and eluted with designed buffer systemcontaining only buffer components used in downstream processes producttrains. In the case of this study, adalimumab bound on MabSelect SuReresin was eluted with 20 mM acetic acid.

Resin

Multimodal media have ligands and/or base matrix with multiplefunctional groups giving a different selectivity compared to traditionalion exchange media. In these examples, the multimodal media having anionexchange and hydrophobic interaction functional groups are shown toremove acidic species as well as other impurities from antibodypreparations.

Capto adhere (GE Healthcare, HiScreen™ prepacked column,Cat#28-9269-81), a strong anion exchanger with multimodal functionality,was evaluated in this study. Its base matrix is a highly cross-linkedagarose with a ligand (N-Benzyl-N-methyl ethanol amine) that exhibitsmany functionalities for interaction, such as ionic interaction,hydrogen bonding and hydrophobic interaction. Those ligands offerdifferent selectivity and hydrophobicity options for proteinseparations.

Capto Adhere Ligand Structure

Methods

Chromatography Method

Pre-packed resin column was used in the following experiments. Thecolumn was equilibrated in a buffer system with appropriate pH andconductivity. The process is illustrated as FIG. 191. The column loadwas prepared from Protein A affinity chromatography. The prepared loadmaterial was filtered and loaded on the column according to the targetload amount (g protein/L resin) as specified followed by washing withthe equilibration buffer and wash buffer similar to equilibration bufferwith volumes as specified. The column Flow Through during load wascollected as a pool and the column Flow Through during wash wascollected separately. The column was then regenerated with 0.1M Aceticacid (pH 3) solution for next cycle use. The cycle A wash pool was mixedwith Protein A eluate to make e antibody material to load at the targetcapacity for the following cycle. pH and conductivity of the combinedpool (Wash pool+Protein A Eluate) was adjusted with 2M Tris and Milli Qwater to achieve designed pH and conductivity. This material was thenfiltered through a 0.45 μm filter (Corning polystyrene).

Buffer Preparation Method

Buffers were prepared targeting specific pH and conductivity by startingwith an anionic component solution (acid) to a target value, andadjusting the solution with the cationic component (base) to achieve theappropriate pH and subsequently adding water to achieve the targetconductivity. For example to prepare a Tris-Acetate buffer solution withpH 7.85 and conductivity of 2.5 mS/cm, a 250 mM acetic acid solution wasadjusted pH to 7.85±0.05 with 3 M Tris solution, the solutionconductivity was then adjusted to 2.5±0.5 mS/cm with addition of water,final solution pH was then confirmed or adjusted to 7.85±0.05 byaddition of 3 M acetic acid solution or 3 M or 2M Tris solution asneeded.

In this study, Tris/Acetate buffer with pH 7.9 and conductivity 2.5mS/cm was used for column equilibration; Tris/Acetate buffer with pH 7.9and conductivity 5.0 mS/cm was used for post load wash buffer.

Capto Adhere load material preparation

Cycle A:

The Protein A eluate was titrated to pH 7.9 with 2M Tris and diluted toconductivity of 2.5 mS/cm with Milli Q water. The prepared material wasthen filtered with 0.22 μm filter before load to column.

Cycle B:

The entire cycle A wash pool was mixed with Protein A eluate to makeenough load for the following cycle. pH and conductivity was adjustedafter mixing with 2M Tris and Milli Q water to achieve pH 7.9 andconductivity of 2.5 mS/cm. This material was then filtered through a0.45 μm filter (Corning polystyrene filter) before load to column.

Cycle C:

The entire cycle B wash pool was mixed with Protein A eluate to makeenough load for the following cycle. pH and conductivity was adjustedafter mixing with 2M Tris and Milli Q water to achieve pH 7.9 andconductivity of 2.5 mS/cm. This material was then filtered through a0.45 μm filter (Corning polystyrene filter) before load to column.

Cycle D:

The entire cycle C wash pool was mixed with Protein A eluate to makeenough load for the following cycle. pH and conductivity was adjustedafter mixing with 2M Tris and Milli Q water to achieve pH 7.9 andconductivity of 2.5 mS/cm. This material was then filtered through a0.45 μm filter (Corning polystyrene filter) before load to column.

AR Reduction and Recovery Calculations

In general, the Flow Through/wash fractions were collected and analyzedwith WCX-10 method for AR levels. By actual or calculated pooling of thefractions the recovery and the corresponding AR levels were calculated.

Analytical Methods

WCX-10 for Adalimumab

The acidic species and other charge variants present in the adalimumabprocess samples were quantified according to the following methods.Cation exchange chromatography was performed on a Dionex ProPac WCX-10,Analytical column 4 mm×250 mm (Dionex, Calif.). An Agilent 1200 HPLCsystem was used as the HPLC. The mobile phases used were 10 mM SodiumPhosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphatedibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binarygradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B:22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm.

Quantitation was based on the relative area percent of detected peaks.The peaks that elute at relative residence time less than a certain timeare together represented as the acidic peaks.

UV spectroscopy A₂₈₀

UV A₂₈₀ was used to determine protein concentrations for the samplespost Protein A elution. The assay was performed on an Agilent UVSpectrophotometer. The protein concentration was determined usingBeer-Lambert's Law, A=εlc, where A is Absorbance, ε is the extinctioncoefficient, 1 is the path length, and c is the concentration. Theabsorbance was taken at 280 nm, the path length was 1 cm, and theextinction coefficients were 1.39 for adalimumab.

Demonstration of Recycle and Continuous Chromatography

In this Example, adalimumab and resin Capto Adhere were chosen. 75 gramsof adalimumab per liter of resin was loaded on a Capto Adhere column ineach cycle and a total four cycles were performed. A single run with 100g/L of load material loaded on the Capto Adhere column was run as areference to compare the AR reduction and mAb recovery. FIG. 192illustrates percent AR in load, flow-through pool (FT), wash pool (wash)of each cycle of the MM process, and the cumulative % AR in overall FT.As shown in FIG. 192, the load % AR in each cycle increased due tohigher % AR in wash pool obtained from previous cycle was re-processed,which led to slight increase of % AR in flow-through pool.

It is clear from FIG. 192 that the AR levels are maintained in thecollected pools in all four cycles, achieving an overall reduction ofapproximately 5%. Thus, it is evident that the recycle mode can maintainthe product AR levels. Table 93 shows the recovery obtained for eachstep and the overall recovery. It is evident that the recycle moderesults in significant improvement in recovery (a 10% increase) when thefour cycles are run, as compared to a single run achieving similarproduct quality. As seen in Table 93, cumulative recovery increases witheach additional cycle. Therefore, additional improvement can be achievedby increasing the number of cycles. Moreover, when comparing theperformance in cycle 1 vs. the performance in cycles 1 to 4(cumulative), it is clear that a 20% increase in recovery can beachieved by using mode of chromatography.

TABLE 93 Acidic Species Reduction and mAb recovery in a Proof-of-Conceptcontinuous MM chromatography Load step amount per Cumulative CumulativeΔ % Cumultive Cycle cycle (g/L) yield (%) % AR AR Δ % AR single 100 627.7 4.1 4.1 run 1 75 52 6.3 5.5 5.5 2 75 62 6.5 6.6 5.3 3 75 67 6.7 7.15.1 4 75 72 6.8 7.1 5.0

Example 12 Storage of AR Reduction

The current invention provides a method for reducing acidic species fora given protein of interest. In this Example, adalimumab was preparedusing a combination of supplementation of arginine and lysine to cellculture as shown in this invention along with AEX and CEX purificationtechnologies, as described herein, to produce a Low-AR and High-ARsample with a final AR of 2.5% and 6.9%, respectively. Both samples wereincubated in a controlled environment at 25° C. and 65% relativehumidity for 10 weeks, and the AR measured every two weeks. FIG. 164shows the growth of AR for each sample over the 10 week incubation. Itis evident from FIG. 164 the growth rate of AR is linear and similarbetween both the Low-AR and High-AR samples. Based on these results thereduced AR material can be stored 3 fold longer before reaching the sameAR level as the High-AR sample. This is a significant utility as thiscan be very beneficial in storage handling and use of the antibody orother proteins for therapeutic use. Moreover, as indicated above, theformation of storage-derived AR can be inhibited when the preparation isstored under particular conditions. For example, an aqueous formulationcan be stored at a particular temperature to partially or completelyinhibit AR formation. In addition, formation or storage-derived AR canbe partially inhibited in an aqueous formulation stored at between about2° C. and 8° C., and completely inhibited when stored at −80° C.Moreover, a low AR composition can be lyophilized to partially orcompletely inhibit the formation of storage-derived AR.

Example 13 Increased Biological Activity of Low AR Compositions

This Example describes the increased efficacy of an exemplary low ARcomposition comprising adalimumab in vivo. The low AR composition usedin this Example was produced as described in Example 8.14, above, usinga CEX reduction method. In particular, the low AR composition used inthis example was produced using a Poros XS column in a Tris/Formatebuffer system at a pH of 7.5. The low AR composition has an AR of 3.1%,wherein the composition comprises 0.1% AR1 and 3.0% AR2. In thisexample, this composition is referred to as the “low AR composition.”

Animal Model for Arthritis

In order to study the efficacy of this low AR adalimumab composition,experiments were carried out in vivo using human TNF-Tg197 mice. TheTNF-Tg197 mouse model is a well recognized mouse model of arthritis usedto test anti-human TNFα treatment modalities. The TNF-Tg197 mouse modelis described in Keffer, J. et al., (1991) EMBO J 10:4025-4031, thecontents of which are incorporated herein by reference. The transgenicmice carrying human TNF gene were developed to study the effects ofexcess TNF production in vivo.

Tg197 mice develop swelling in the ankle joints of both hind paws andimpaired movement, which is very similar to human rheumatoid arthritis.Clinical signs of disease in Tg197 mice start at 4 weeks of age andinclude slower weight gain, joint distortion and swelling, jointdeformation and ankylosis and impaired movement. Histopathologicalanalysis reveals hyperplasia of synovial membrane, leukocyteinfiltration at around 3 weeks of age, and then pannus formation,articular cartilage destruction and massive production of fibrous tissueat advanced stage of disease at 9-11 weeks of age. This model has beenused in the development of anti-TNFα biologics, including adalimumab.

Methods

Groups of mice (6 males and 6 females), were administered one of thefollowing adalimumab formulations: low AR composition (group 5), lowhost cell protein (HCP) composition (group 7), AR1 composition(containing only AR1 acidic variants) (group 8), and Lys-1/2 composition(containing only Lys 1 and Lys 2 variants) (group 9). These compositions(fractions) are shown in the chromatograph in FIG. 193. Another group ofmice was administered a control composition, also referred to as the“control AR composition,” or “normal” composition, which containsadalimumab with unmodified AR levels and unmodified Lys variants. Aplacebo group, comprising 6 mice, was also included.

Each composition, including the control AR composition, was administeredto the mice in each group beginning with a tolerizing dose of adalimumabat age 1 week, and followed by additional weekly dosages of 1 mg/kg for10 weeks. From weeks 2.5 through weeks 13.5, weekly measurements ofweight and arthritic scores were taken and weekly serum collection wasmade. In addition, at the end of the study, tissue samples from perfusedmice were obtained and analyzed. The following tissues were harvestedfor testing drug levels, anti-drug antibodies (ADA), and complexed andfree TNF levels: front paws, inguinal, popliteal and mesenteric lymphnodes, spleen, tail (for skin sample), knees. The femur and spinetissues were harvested for micro-CT scanning.

Results

As shown in FIG. 194A, the mice receiving the low AR composition had thelowest arthritic scores of all of the compositions tested, including thecontrol AR composition, indicating increased efficacy in the treatmentof arthritis. Furthermore, as shown in FIG. 194B, the mice administeredthe low AR composition exhibited an average weight gain that wascomparable to the control composition, indicating safety of the low ARcomposition and a lack of adverse effects of the low AR composition thatimpact weight gain and growth of the mice.

As shown in FIG. 195, during the 12-13 week treatment period of themice, the low AR composition provided the best protection againstdevelopment of arthritis in the mice, as measured by arthritic scores,as compared to the other compositions tested. The Lys-1/2 compositionwas the next most effective. The AR1 composition offered the leastprotection against development of arthritic scores, and it was lessprotective than the control AR composition.

Serum levels of ADA and drug levels were measured from 3 to 14 weeks ofage. As shown in FIG. 196B, animals administered the low AR compositionexhibited low average levels of ADA across the time frame measured. Inaddition, animals administered the low AR composition exhibited drugserum levels comparable to the control (FIG. 196A), indicating that alack of presence of the drug in the serum was not responsible for thelow levels of serum ADA.

As set forth in FIG. 197, cumulative serum concentration values (PK)during the ten week treatment period was highest for the animalsadministered the low AR composition and lowest for the animalsadministered the AR1 composition. The Lys-1/2 composition was the nextbest following the low AR composition, and was higher than the ARcontrol composition. As also shown in FIG. 197, the highest ADA titerswere observed for animals administered the AR1 composition and thelowest for animals administered the low AR composition.

Furthermore, complexed TNF levels show that cumulative serumconcentration values during the ten week treatment period were highestfor animals administered the control AR composition and lowest for theanimals administered the AR1 composition (FIG. 198). Cumulative serumconcentration values for the low AR composition were slightly less thanthe levels of the control AR composition.

A histopathology evaluation of the joints of the mice indicated that thebest protection was afforded by the low AR composition and the Lys-1/2composition, indicating that the low AR composition and the Lys-1/2composition protect against the formation of arthritis in the joints invivo. As shown in FIG. 199, the low AR composition protected againstcell infiltration, synovial proliferation, proteoglycan loss, cartilagedestruction, and bone erosion more effectively than the othercompositions, including the control AR composition. Protection by theAR1 composition was lower than the control AR composition, indicating adetrimental effect by AR1 with respect to joint damage.

FIGS. 200A-D illustrate the average drug (PK) levels for various tissues(paw, lymph node, spleen, skin, knee and serum) for the low ARcomposition, the control AR composition, the AR1 composition, and theLys-1/2 composition. As shown therein, animals administered the low ARcomposition had drug levels as high or higher than animals administeredthe other compositions tested.

FIG. 201A-D illustrates average ADA levels in the same tissues for thesame compositions (the low AR composition, the control AR composition,the AR1 composition, and the Lys-1/2 composition). As shown in FIG.201A-D, for the low AR composition, the highest ADA concentrations arepresent in the paws (which corresponds to the location of the highestlevels of inflammation in the animals), and the serum.

FIGS. 202A-D and 203A-D show the results of a micro CT analysis ofspines and femurs obtained from the transgenic mice at the end of thestudy that were administered low AR composition, control AR composition,AR1 composition, Lys-1/2 composition, as well as naïve, (control) andplacebo. Samples were analyzed for L5 vertebra bone volume, L5 vertebratrabecular number, L5 vertebra trabecular thickness, and L5 vertebratrabecular space. As shown in FIGS. 202A-D and 203A-D, the low ARcomposition and the Lys-1/2 composition resulted in greater bone volume,trabecular number, trabecular thickness and trabecular space, ascompared to the control (normal) AR composition.

FIGS. 204A-D show additional results of a micro CT analysis of spinesand femurs obtained from the transgenic mice at the end of the studythat were administered low AR composition, control AR composition, AR1composition, Lys-1/2 composition, as well as naïve (control), andplacebo. Samples were analyzed for trabecula bone volume at the femoralmetaphysis, trabecular number at the femoral metaphysis, trabecularthickness at the femoral metaphysis, and trabecular separation at thefemoral metaphysis. As shown in FIGS. 204A-D, the low AR compositionresulted in greater trabecula bone volume at the femoral metaphysis,trabecular number at the femoral metaphysis, and trabecular thickness atthe femoral metaphysis, as compared to the control (normal) ARcomposition.

Furthermore, FIGS. 205 and 206 show actual micro CT images of the spineand femur, respectively, from each of six groups of mice administeredthe following compositions: naïve, vehicle (control), low AR composition(group 5), low host cell protein (HCP) composition (group 7), AR1composition (containing only AR1 acidic variants) (group 8), and Lys-1/2composition (containing only Lys 1 and Lys 2 variants) (group 9). Asseen in both the spine and the femur, the low AR composition (group 5),provided protection from bone erosion, as compared to the vehicle, asthere is less bone erosion visible in the group 5 image as compared tothe vehicle.

The results of these experiments demonstrate that a weekly dose of 1mg/kg adalimumab in TNF-Tg197 mice provides protection from arthritisdevelopment as measured by arthritic scores and histopathology scores(radiologic damage involving cartilage and bone as well as localinflammation) in the TNF-Tg197 mouse model. Thus, the control ARcomposition, with normal level of AR variants, was efficacious at acertain level.

Formulations containing either the low AR formulation or the Lys-1/2composition provided greatest protection, as compared to the control ARgroup, from development of arthritis as measured by arthritic scores andhistopathology scores, and showed increased efficacy, as compared to thecontrol AR group, in all parameters tested including cell infiltration,synovial proliferation, proteoglycan loss, cartilage destruction, andbone erosion. Accordingly, the low AR composition and the Lys-1/2composition have increased efficacy in the treatment and prevention ofarthritis as compared to the control AR composition.

The adalimumab AR1 composition was less efficacious than the normal ARcontaining adalimumab control group in all aspects interrogated in thecurrent study: less weight gain, higher arthritic scores, and higherhistopathology scores in the joints, indicating a detrimental effectexerted by AR1.

Noteworthy differences were observed in serum levels of the variousformulations include the following: the animals treated with the AR1composition had the lowest concentration of adalimumab as compared tothe other groups, and the animals treated with the low AR compositionhad the highest concentration of adalimumab as compared to the othergroups. The AR1 composition also had the highest titers of ADA in serum.

* * *

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

The contents of all cited references, including literature references,issued patents, and published patent applications, as cited throughoutthis application are hereby expressly incorporated herein by reference.It should further be understood that the contents of all the figures andtables attached hereto are expressly incorporated herein by reference.The entire contents of the following applications are also expresslyincorporated herein by reference: U.S. Provisional Patent Application61/893,123, entitled “STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OFMAKING SAME”, filed on Oct. 18, 2013; U.S. Provisional Application Ser.No. 61/892,833, entitled “LOW ACIDIC SPECIES COMPOSITIONS AND METHODSFOR PRODUCING THE SAME USING DISPLACEMENT CHROMATOGRAPHY”, filed on Oct.18, 2013; U.S. Provisional Patent Application 61/892,710, entitled“MUTATED ANTI-TNFα ANTIBODIES AND METHODS OF THEIR USE”, filed on Oct.18, 2013; U.S. Provisional Patent Application 61/893,088, entitled“MODULATED LYSINE VARIANT SPECIES AND METHODS FOR PRODUCING AND USINGTHE SAME”, filed on Oct. 18, 2013; and U.S. Provisional PatentApplication 61/893,131, entitled “PURIFICATION OF PROTEINS USINGHYDROPHOBIC INTERACTION CHROMATOGRAPHY”, filed on Oct. 18, 2013.

The invention claimed is:
 1. A method for producing a compositioncomprising an immunoglobulin comprising the 6 CDR domains of adalimumab,the method comprising: contacting a first sample comprising theimmunoglobulin, wherein the sample comprises more than 10% total acidicspecies of the immunoglobulin, to a first chromatography media in thepresence of a loading buffer to produce a first chromatography sample,wherein the first chromatography media is selected from the groupconsisting of an ion exchange chromatography media, an affinitychromatography media and a hydrophobic interaction chromatography (HIC)media, wherein the first chromatography sample comprises a compositionof the immunoglobulin comprising less than 10% total acidic species ofthe immunoglobulin, wherein the acidic species of the immunoglobulincorrespond to the peaks that elute earlier than the main peak in aWCX-10 HPLC chromatogram of the immunoglobulin, wherein the WCX-10 HPLCchromatogram is generated using a first mobile phase of 10 mM SodiumPhosphate dibasic (pH 7.5) and a second mobile phase of 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and wherein theWCX-10 HPLC chromatogram is generated using detection at 280 nm.
 2. Themethod of claim 1, wherein producing the first chromatography samplecomprises washing the first chromatography media with a wash buffer. 3.The method of claim 2, wherein the wash buffer is the same as theloading buffer.
 4. The method of claim 1, wherein the firstchromatography sample is a flow through chromatography sample whichcomprises less than 10% total acidic species of the immunoglobulin. 5.The method of claim 1, wherein producing the first chromatography samplecomprises eluting the first chromatography sample, thereby producing afirst eluted sample which comprises less than 10% total acidic speciesof the immunoglobulin.
 6. The method of claim 5, further comprisingcontacting the first eluted sample to a second chromatography media andeluting the sample, thereby producing a second eluted sample, whereinthe second chromatography media is selected from the group consisting ofan ion exchange chromatography media, an affinity chromatography mediaand a hydrophobic interaction chromatography (HIC) media.
 7. The methodof claim 6, further comprising contacting the second eluted sample to athird chromatography media and eluting the sample, thereby producing athird eluted sample, wherein the third chromatography media is selectedfrom the group consisting of an ion exchange chromatography media, anaffinity chromatography media and a hydrophobic interactionchromatography (HIC) media.
 8. The method of claim 7, wherein the firstchromatography media is an affinity chromatography media, the secondchromatography media is an ion exchange chromatography media and thethird chromatography media is a hydrophobic interaction chromatography(HIC) media.
 9. The method of claim 7, wherein the first chromatographymedia is an affinity chromatography media, the second chromatographymedia is an ion exchange chromatography media and the thirdchromatography media is an ion exchange chromatography media.
 10. Themethod of claim 9, wherein the affinity chromatography media is aprotein A chromatography media, the ion exchange chromatography mediaused as the second chromatography media is an anion exchange (AEX)chromatography media and the ion exchange chromatography media used asthe third chromatography media is a cation exchange (CEX) chromatographymedia.
 11. The method of claim 1, wherein the first chromatography mediais an ion exchange chromatography media selected from the groupconsisting of an anion exchange (AEX) chromatography adsorbent material,a cation exchange (CEX) chromatography adsorbent material, a cationexchange mixed mode media, and an anion exchange mixed mode media. 12.The method of claim 11, wherein the cation exchange (CEX) adsorbentmaterial is selected from the group consisting of a CEX resin and a CEXmembrane adsorber.
 13. The method of claim 11, wherein the anionexchange (AEX) adsorbent material is selected from the group consistingof an AEX resin and an AEX membrane adsorber.
 14. The method of claim 1,wherein the first chromatography media is an affinity chromatographymedia.
 15. The method of claim 14, wherein the affinity chromatographymedia is a Protein A chromatography media.
 16. The method of claim 1,wherein the first chromatography media is a hydrophobic interactionchromatography (HIC) media.
 17. The method of claim 1, wherein the firstchromatography media is a mixed mode media comprising ion exchange andhydrophobic interaction functional groups.
 18. The method of claim 1,wherein the first chromatography media is a CEX adsorbent material or amixed mode media, and the pH of the loading buffer is lower than theisoelectric point of adalimumab.
 19. The method of claim 1, wherein theimmunoglobulin is adalimumab and wherein the total acidic species ofadalimumab comprise a first acidic region (AR1) and a second acidicregion (AR2).
 20. The composition of claim 19, wherein the first acidicregion (AR1) and the second acidic region (AR2) comprise chargevariants, structure variants and fragmentation variants.
 21. Thecomposition of claim 20, wherein the charge variants comprise one ormore of deamidation variants, glycation variants, afucosylationvariants, MGO variants or citric acid variants, the structure variantscomprise one or more of glycosylation variants or acetonation variants,and the fragmentation variants comprise one or more of Fab fragmentvariants, C-terminal truncation variants or variants missing a heavychain variable domain.
 22. The method of claim 1, wherein thecomposition comprises 9% or less, 8% or less, 7% or less, 6% or less, 5%or less, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% orless, or 2% or less total acidic species of the immunoglobulincomprising the 6 CDR domains of adalimumab.
 23. The method of claim 1,wherein the immunoglobulin is adalimumab.
 24. The method of claim 1,wherein the composition comprises 1.4% to 10% total acidic species ofthe immunoglobulin comprising the 6 CDR domains of adalimumab.
 25. Themethod of claim 1, wherein the composition comprises 1.4% or less totalacidic species of the immunoglobulin comprising the 6 CDR domains ofadalimumab.
 26. A method for producing a composition comprising animmunoglobulin comprising the 6 CDR domains of adalimumab, the methodcomprising: (a) contacting a first sample comprising the immunoglobulin,wherein the sample comprises more than 10% total acidic species of theimmunoglobulin, to an affinity chromatography media in a first loadingbuffer, and eluting a first eluted sample from the affinitychromatography media; (b) contacting the first eluted sample to a secondchromatography media in a second loading buffer and eluting a secondeluted sample from the second chromatography media; and (c) contactingthe second eluted sample to a third chromatography media in a thirdloading buffer and eluting a third eluted sample from the thirdchromatography media; wherein the third eluted sample comprises acomposition of the immunoglobulin comprising less than 10% total acidicspecies of the immunoglobulin, wherein the acidic species of theimmunoglobulin correspond to the peaks that elute earlier than the mainpeak in a WCX-10 HPLC chromatogram of the immunoglobulin, wherein theWCX-10 HPLC chromatogram is generated using a first mobile phase of 10mM Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10 mMSodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and whereinthe WCX-10 HPLC chromatogram is generated using detection at 280 nm. 27.The method of claim 26, wherein the second chromatography media isselected from the group consisting of an anion exchange (AEX)chromatography adsorbent material, a cation exchange (CEX)chromatography adsorbent material, a mixed mode media, a cation exchangemixed mode media, and an anion exchange mixed mode media.
 28. The methodof claim 26, wherein the third chromatography media is selected from thegroup consisting of an anion exchange (AEX) chromatography adsorbentmaterial a cation exchange (CEX) chromatography adsorbent material, amixed mode media, a cation exchange mixed mode media, and an anionexchange mixed mode media.
 29. The method of claim 26, wherein thesecond loading buffer, the third loading buffer, or both the second andthird loading buffers are a Tris/Formate buffer.
 30. The method of claim26, wherein the composition comprises 1.4% to 10% total acidic speciesof the immunoglobulin comprising the 6 CDR domains of adalimumab. 31.The method of claim 26, wherein the composition comprises 1.4% or lesstotal acidic species of the immunoglobulin comprising the 6 CDR domainsof adalimumab.
 32. The method of claim 26, wherein the compositioncomprises 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, or 2%or less total acidic species of the immunoglobulin comprising the 6 CDRdomains of adalimumab.
 33. The method of claim 26, wherein theimmunoglobulin is adalimumab.
 34. A method for producing a compositioncomprising an immunoglobulin comprising the 6 CDR domains of adalimumab,the method comprising: (a) contacting a first sample comprising theimmunoglobulin, wherein the sample comprises more than 10% total acidicspecies of the immunoglobulin, to a protein A chromatography media in afirst loading buffer, and eluting a first eluted sample from the proteinA chromatography media; (b) contacting the first eluted sample to ananion exchange (AEX) chromatography media in a second loading buffer andeluting a second eluted sample from the AEX chromatography media; and(c) contacting the second eluted sample to a cation exchange (CEX)chromatography media in a third loading buffer and eluting a thirdeluted sample from the CEX chromatography media; wherein the thirdeluted sample comprises a composition of the immunoglobulin comprisingless than 10% total acidic species of the immunoglobulin, wherein theacidic species of the immunoglobulin correspond to the peaks that eluteearlier than the main peak in a WCX-10 HPLC chromatogram of theimmunoglobulin, wherein the WCX-10 HPLC chromatogram is generated usinga first mobile phase of 10 mM Sodium Phosphate dibasic (pH 7.5) and asecond mobile phase of 10 mM Sodium Phosphate dibasic, 500 mM SodiumChloride (pH 5.5), and wherein the WCX-10 HPLC chromatogram is generatedusing detection at 280 nm.
 35. The method of claim 34, wherein theimmunoglobulin is adalimumab.
 36. The method of claim 34, wherein thecomposition comprises 1.4% to 10% total acidic species of theimmunoglobulin comprising the 6 CDR domains of adalimumab.
 37. Themethod of claim 34, wherein the composition comprises 1.4% or less totalacidic species of the immunoglobulin comprising the 6 CDR domains ofadalimumab.
 38. The method of claim 34, wherein the compositioncomprises 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, or 2%or less total acidic species of the immunoglobulin comprising the 6 CDRdomains of adalimumab.
 39. A method for producing a compositioncomprising adalimumab, the method comprising: (a) contacting a firstsample comprising adalimumab, wherein the sample comprises more than 10%total acidic species of adalimumab, to a protein A chromatography mediain a first loading buffer, and eluting a first eluted sample from theprotein A chromatography media; (b) contacting the first eluted sampleto an anion exchange (AEX) chromatography media in a second loadingbuffer and eluting a second eluted sample from the AEX chromatographymedia; and (c) contacting the second eluted sample to a cation exchange(CEX) chromatography media in a third loading buffer and eluting a thirdeluted sample from the CEX chromatography media; wherein the thirdeluted sample comprises a composition of adalimumab comprising less than10% total acidic species of adalimumab, wherein the acidic species ofadalimumab correspond to the peaks that elute earlier than the main peakin a WCX-10 HPLC chromatogram of adalimumab, wherein the WCX-10 HPLCchromatogram is generated using a first mobile phase of 10 mM SodiumPhosphate dibasic (pH 7.5) and a second mobile phase of 10 mM SodiumPhosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and wherein theWCX-10 HPLC chromatogram is generated using detection at 280 nm.
 40. Themethod of claim 39, wherein the composition comprises 1.4% to 10% totalacidic species of adalimumab.
 41. The method of claim 39, wherein thecomposition comprises 1.4% or less total acidic species of adalimumab.42. The method of claim 39, wherein the composition comprises 9% orless, 8% or less, 7% or less, 6% or less, 5% or less, 4.5% or less, 4%or less, 3.5% or less, 3% or less, 2.5% or less, or 2% or less totalacidic species of adalimumab.